Respiratory virus nucleic acid vaccines

ABSTRACT

Provided herein, in some embodiments, are vaccines (and vaccination methods) that include a ribonucleic acid (RNA) polynucleotide encoding a human metapneumovims (hMPV) F protein and a RNA polynucleotide encoding a human parainfluenza virus 3 (hPrV3) F protein.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application No. 62/431,775, filed Dec. 8, 2016, which isincorporated by reference herein in its entirety.

BACKGROUND

Human metapneumovirus (hMPV), discovered in 2001, is a common cause ofupper and lower respiratory infections. Although often mild, this viruscan be serious and life-threatening in high-risk groups, such aschildren under the age of 5 years, elderly adults over the age of 65years, and adults with underlying disease (e.g., Chronic ObstructivePulmonary Disease (COPD), asthma, congestive heart failure, ordiabetes). In healthy adults over the age of 65 years, the annualincidence rate of hMPV infection is 1.2/1,000, and 38% of these elderlyadults require medical care (compared to 9% of young adults). hMPVinfection in elderly adults is a common cause of respiratory infectionoutbreaks (attack rates 13-30%) in residential care facilities, andhospitalization rates are higher than those for influenza infectionoccurring in healthy adults over the age of 50 years. For individualswho have an underlying pulmonary disease, hMPV infection is associatewith exacerbations of the disease (e.g., COPD), and individuals aretwice as likely to have symptomatic disease and requirement for medicalcare. In immunocompromised individuals, hMPV is responsible for 6% oftotal respiratory infections in lung transplants and 3% of lowerrespiratory infections associated with stem cell transplant. hMPVinfection is also thought to be associated with acute graft rejection.

Likewise, human parainfluenza virus 3 (hPIV3) is also a common cause ofupper and lower respiratory infections. This serotype is the mostpathogenic of the four PIV serotypes. Infection of hPIV3 in high riskgroups can result in serious lower respiratory infections, includingbronchiolitis and/or pneumonia.

SUMMARY

Provided herein, in some embodiments, are ribodeoxynucleic acid (RNA)(e.g., mRNA) vaccine compositions and methods for preventing and/ortreating lower respiratory human metapneumovirus (hMPV) and humanparainfluenza virus 3 (hPIV3) infections, for example, in infants andyoung adults, in elderly adults, and in those with underlyingrespiratory diseases. The hMPV/hPIV3 vaccines of the present disclosureproduce proteins inside cells, which in turn are secreted or are activeintracellularly. In some instances, RNA (e.g., mRNA) vaccines producemuch larger antibody titers and produce immune responses earlier,relative to current anti-viral therapeutic treatments. Without beingbound by theory, it is believed that the hMPV/hPIV3 RNA vaccines, asprovided here, are better designed to produce the appropriate proteinconformation upon translation, as the RNA vaccines co-opt naturalcellular machinery. Unlike traditional vaccines, which are manufacturedex vivo and may trigger unwanted cellular responses, RNA vaccines arepresented to the cellular system in a more native fashion.

Surprisingly, administration of the hMPV/hPIV3 vaccine of the disclosureinduces high neutralizing antibody titers and reduced viral load, butdoes not result in alveolitis or interstitial pneumonia.

Some embodiments of the present disclosure provide a vaccine, comprising(a) a ribonucleic acid (RNA) polynucleotide encoding a humanmetapneumovirus (hMPV) antigenic polypeptide comprising the amino acidsequence identified by SEQ ID NO:7 or an amino acid sequence that is atleast 95% identical to the amino acid sequence identified by SEQ IDNO:7, and (b) a RNA polynucleotide encoding human parainfluenza virus 3(hPIV3) antigenic polypeptide comprising the amino acid sequenceidentified by SEQ ID NO:8 or an amino acid sequence that is at least 95%identical to the amino acid sequence identified by SEQ ID NO:8.

Some embodiments of the present disclosure provide a vaccine, comprising(a) a RNA polynucleotide comprising a nucleic acid sequence identifiedby SEQ ID NO:4 encoding a hMPV antigenic polypeptide or an amino acidsequence that is at least 95% identical to the amino acid sequenceidentified by SEQ ID NO:4 encoding a hMPV antigenic polypeptide and (b)a RNA polynucleotide comprising a nucleic acid sequence identified bySEQ ID NO:5 encoding a hPIV3 antigenic polypeptide or an amino acidsequence that is at least 95% identical to the amino acid sequenceidentified by SEQ ID NO:5 encoding a hPIV3 antigenic polypeptide.

In some embodiments, the RNA polynucleotides of (a) and (b) areformulated in a lipid nanoparticle comprising a cationic lipid, aPEG-modified lipid, a sterol and a non-cationic lipid.

In some embodiments, the vaccine comprises (a) a RNA polynucleotidecomprising the nucleic acid sequence identified by SEQ ID NO:4 encodinga hMPV antigenic polypeptide and (b) a RNA polynucleotide comprising thenucleic acid sequence identified by SEQ ID NO:5 encoding a hPIV3antigenic polypeptide. In some embodiments, the vaccine includes a 5′UTR, a 3′ UTR, a polyA tail (e.g., 100 nucleotides), a cap (e.g.,7mG(5′)ppp(5′)NlmpNp), or any combination of two or more of theforegoing components. In some embodiments, the 5′ UTR comprises asequence identified by SEQ ID NO:12. In some embodiments, the 3′ UTRcomprises a sequence identified by SEQ ID NO:13. Other known UTRsequences may be used. In some embodiments, the RNA polynucleotide of(a) and/or (b) is chemically modified (e.g., comprises1-methylpseudouridine modifications). In some embodiments, the vaccinecomprises (a) a RNA polynucleotide comprising the nucleic acid sequenceidentified by SEQ ID NO:14 encoding a hMPV antigenic polypeptide and (b)a RNA polynucleotide comprising the nucleic acid sequence identified bySEQ ID NO:15 encoding a hPIV3 antigenic polypeptide. In someembodiments, the vaccine is formulated in a lipid nanoparticle, such asa cationic lipid nanoparticles. In some embodiments, the cationic lipidnanoparticle comprises a mixture of: Compound 1 lipids;1,2-dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG2000-DMG);1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and cholesterol. Insome embodiments, the vaccine comprises a 12.5 μg-200 12.5 μg dose ofthe RNA polynucleotide of (a) and a 12.5 μg-200 12.5 μg dose of the RNApolynucleotide of (b).

In some embodiments, the vaccine comprises (a) a RNA polynucleotidecomprising a the nucleic acid sequence identified by SEQ ID NO:14encoding a hMPV antigenic polypeptide and (b) a RNA polynucleotidecomprising the nucleic acid sequence identified by SEQ ID NO:15 encodinga hPIV3 antigenic polypeptide, wherein the RNA polynucleotide of (a) andthe RNA polynucleotide of (b) are co-formulated in a cationic lipidnanoparticle that comprises a mixture of: Compound 1 lipids;1,2-dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG2000-DMG);1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and cholesterol. Insome embodiments, the RNA polynucleotide of (a) and the RNApolynucleotide of (b) are formulated in separate cationic lipidnanoparticles that comprises a mixture of: Compound 1 lipids;1,2-dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG2000-DMG);1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and cholesterol. Insome embodiments, the vaccine comprises a 12.5 μg-200 12.5 μg dose ofthe RNA polynucleotide of (a) and a 12.5 μg-200 12.5 μg dose of the RNApolynucleotide of (b).

In some embodiments, hMPV and/or hPIV3 viral load is undetectable insubjects after challenge with the virus(es) following administration ofless than three doses of the vaccine. In some embodiments, hMPV and/orhPIV3 viral load is undetectable in subjects after challenge with thevirus(es) following administration of two doses of the vaccine. In someembodiments, hMPV and/or hPIV3 viral load is undetectable in subjectsafter challenge with the virus(es) following administration of a singledose of the vaccine.

In some embodiments, the anti-hPIV3 neutralizing antibody titer producedin a subject following administration of a dose of the vaccine is atleast 3-fold higher than the anti-hPIV3 neutralizing antibody titerproduced in a subject following administration of a comparable dose of avaccine comprising mRNA encoding hPIV3 HN protein.

In some embodiments, the vaccine provides an effective immune responseagainst both hMPV and hPIV3.

In some embodiments, the anti-hPIV3 and/or anti-hMPV neutralizingantibody titer produced in cotton rats following administration of thevaccine is at least 9 on a log base 2 scale, as measured at the 60%reduction end point of virus control.

In some embodiments, the hMPV viral load in the lung and/or nose isbelow the limit of quantification in subjects following administrationof the vaccine and challenge with hMPV.

In some embodiments, the hPIV3 viral load in the lung and/or nose isbelow the limit of quantification in subjects following administrationof the vaccine and challenge with the hPIV3.

In some embodiments, a subject administered the vaccine does not exhibitsymptoms of vaccine-enhanced respiratory disease (e.g., alveolitis(cells within the alveolar spaces) or interstitial pneumonia(inflammatory cell infiltration and thickening of alveolar walls)).

In some embodiments, the neutralizing antibody titer against hMPV in asubject following administration of a second dose of the vaccine isincreased by 8-10 fold at 14 days post-administration of the seconddose.

In some embodiments, the neutralizing antibody titer against hPIV3 in asubject following administration of a second dose of the vaccine isincreased by 4-10 fold at 14 days post-administration of the seconddose.

Other embodiments of the present disclosure provide a vaccine,comprising a ribonucleic acid (RNA) polynucleotide encoding a humanmetapneumovirus (hMPV) antigenic polypeptide comprising the amino acidsequence identified by SEQ ID NO:7, wherein the hMPV RNA polynucleotideis formulated in a lipid nanoparticle comprising a cationic lipid, aPEG-modified lipid, a sterol and a non-cationic lipid.

Yet other embodiments of the present disclosure provide a vaccine,comprising a ribonucleic acid (RNA) polynucleotide encoding humanparainfluenza virus 3 (hPIV3) antigenic polypeptide comprising the aminoacid sequence identified by SEQ ID NO:8, wherein the hPIV3 RNApolynucleotide is formulated in a lipid nanoparticle comprising acationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid.

In some embodiments, the vaccine further comprises a RNA polynucleotideencoding a respiratory syncytial virus (RSV) antigenic polypeptide.

In some embodiments, the cationic lipid is an ionizable lipid.

In some embodiments, the sterol is a cholesterol.

In some embodiments, the non-cationic lipid is a neutral lipid.

In some embodiments, the cationic lipid comprises a compound of FormulaI. In some embodiments, the compound of Formula I is Compound 3, 18, 20,25, 26, 29, 30, 60, 108-112, or 122. In some embodiments, the compoundof Formula I is Compound 25.

In some embodiments, the lipid nanoparticle comprises a molar ratio ofabout 20-60% cationic lipid, 0.5-15% PEG-modified lipid, 25-55% sterol,and 25% non-cationic lipid.

In some embodiments, the lipid nanoparticle has a polydispersity valueof less than 0.4.

In some embodiments, the lipid nanoparticle has a net neutral charge ata neutral pH value.

In some embodiments, the vaccine is formulated in an effective amount toprevent or treat a lower respiratory hMPV/hPIV3 infection in a subject.

In some embodiments, the effective amount is 5 μg-100 μg of the RNApolynucleotide encoding hMPV antigenic polypeptide and/or 5 μg -100 μgof the RNA polynucleotide encoding hPIV3 antigenic polypeptide. In someembodiments, the effective amount is 12.5 μg of the RNA polynucleotideencoding hMPV antigenic polypeptide and/or 12.5 μg of the RNApolynucleotide encoding hPIV3 antigenic polypeptide. In someembodiments, the effective amount is 25 μg of the RNA polynucleotideencoding hMPV antigenic polypeptide and/or 25 μg of the RNApolynucleotide encoding hPIV3 antigenic polypeptide. In someembodiments, the effective amount is 50 μg of the RNA polynucleotideencoding hMPV antigenic polypeptide and/or 50 μg of the RNApolynucleotide encoding hPIV3 antigenic polypeptide.

In some embodiments, the RNA polynucleotide encoding hMPV antigenicpolypeptide and/or the RNA polynucleotide encoding hPIV3 antigenicpolypeptide comprises at least one chemical modification.

In some embodiments, the chemical modification is selected frompseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine,2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine.

In some embodiments, the chemical modification is in the 5-position ofthe uracil. In some embodiments, the chemical modification is aN1-methylpseudouridine or N1-ethylpseudouridine.

In some embodiments, at least 80% of the uracil in the open readingframe have a chemical modification. In some embodiments, at least 90% ofthe uracil in the open reading frame have a chemical modification. Insome embodiments, 100% of the uracil in the open reading frame have achemical modification.

In some embodiments, the RNA polynucleotide encoding hMPV antigenicpolypeptide and/or the RNA polynucleotide encoding hPIV3 antigenicpolypeptide further encodes at least one 5′ terminal cap. In someembodiments, the 5′ terminal cap is 7mG(5′)ppp(5′)NlmpNp.

In some embodiments, the vaccine further comprises an adjuvant. In someembodiments, the adjuvant is a flagellin protein or peptide.

Some embodiments provide a method of preventing a lower respiratoryhuman metapneumovirus and/or human parainfluenza virus 3 (hMPV/hPIV3)infection in elderly subjects, comprising administering to a subject whois 65 years of age or older the vaccine of the present disclosure in aneffective amount to prevent a lower respiratory hMPV/hPIV3 infection inthe subject.

Other embodiments provide a method of treating a lower respiratory humanmetapneumovirus and/or human parainfluenza virus 3 (hMPV/hPIV3)infection in elderly subjects, comprising administering to a subject whois 65 years of age or older and is infected with hMPV/hPIV3 the vaccineof the present disclosure in an effective amount to treat a lowerrespiratory hMPV/hPIV3 infection in the subject.

Yet other embodiments provide a method of preventing a lower respiratoryhuman metapneumovirus and/or human parainfluenza virus 3 (hMPV/hPIV3)infection in a child, comprising administering to a subject who is 5years of age or younger the vaccine of the present disclosure in aneffective amount to prevent a lower respiratory hMPV/hPIV3 infection inthe subject.

Still other embodiments provide a method of treating a lower respiratoryhuman metapneumovirus and/or human parainfluenza virus 3 (hMPV/hPIV3)infection in a child, comprising administering to a subject who is 5years of age or younger and is infected with hMPV/hPIV3 the vaccine ofthe present disclosure in an effective amount to treat a lowerrespiratory hMPV/hPIV3 infection in the subject. In some embodiments,the subject is between 6 and 12 months of age.

Further embodiments provide a method of preventing a lower respiratoryhuman metapneumovirus and/or human parainfluenza virus 3 (hMPV/hPIV3)infection in subjects having a pulmonary disease, comprisingadministering to a subject having a pulmonary disease the vaccine of thepresent disclosure in an effective amount to prevent a lower respiratoryhMPV/hPIV3 infection in the subject.

Still further embodiments provide a method of treating a lowerrespiratory human metapneumovirus and/or human parainfluenza virus 3(hMPV/hPIV3) infection in subjects having a pulmonary disease,comprising administering to a subject having a pulmonary condition thevaccine of the present disclosure in an effective amount to treat alower respiratory hMPV/hPIV3 infection in the subject. In someembodiments, the pulmonary condition is associated with is ChronicObstructive Pulmonary Disease (COPD), asthma, congestive heart failureor diabetes, or any combination thereof.

Some embodiments provide a method of preventing a lower respiratoryhuman metapneumovirus and/or human parainfluenza virus 3 (hMPV/hPIV3)infection in immunocompromised subjects, comprising administering to animmunocompromised subject the vaccine of the present disclosure in aneffective amount to prevent a lower respiratory hMPV/hPIV3 infection inthe subject.

Other embodiments provide a method of treating a lower respiratory humanmetapneumovirus and/or human parainfluenza virus 3 (hMPV/hPIV3)infection in immunocompromised subjects, comprising administering to animmunocompromised subject the vaccine of the present disclosure in aneffective amount to treat a lower respiratory hMPV/hPIV3 infection inthe subject.

In some embodiments, a single dose of the vaccine is administered to thesubject. In some embodiments, a booster dose of the vaccine isadministered to the subject.

In some embodiments, the efficacy of the vaccine against the hMPV/hPIV3infection is at least 50% following administration of the booster doseof the vaccine. In some embodiments, the efficacy of the vaccine againstthe hMPV/hPIV3 infection is at least 60% following administration of thebooster dose of the vaccine.

In some embodiments, the efficacy of the vaccine against the hMPV and/orhPIV3 infection is at least 65% following administration of a singledose of the vaccine. In some embodiments, the efficacy of the vaccineagainst the hMPV and/or hPIV3 infection is at least 70% followingadministration of a single dose of the vaccine. In some embodiments, theefficacy of the vaccine against the hMPV and/or hPIV3 infection is atleast 75% following administration of a single dose of the vaccine.

In some embodiments, the vaccine immunizes the subject againsthMPV/hPIV3 for up to 2 years. In some embodiments, the vaccine immunizesthe subject against hMPV/hPIV3 for more than 2 years.

Also provided herein is a vaccine, comprising (a) 12.5 μg-200 μg a humanmetapneumovirus (hMPV) ribonucleic acid (RNA) polynucleotide comprisingthe nucleic acid sequence identified by SEQ ID NO:4, and (b) 12.5 μg-200μg a human parainfluenza virus 3 (hPIV3) RNA polynucleotide comprisingthe nucleic acid sequence identified by SEQ ID NO:5, wherein the RNApolynucleotides of (a) and (b) are formulated in a lipid nanoparticlecomprising a Compound 25 of Formula (I), a PEG-modified lipid, a steroland a non-cationic lipid. In some embodiments, the efficacy of thevaccine against the hMPV/hPIV3 infection is at least 50% followingadministration of the booster dose of the vaccine. In some embodiments,the efficacy of the vaccine against the hMPV and/or hPIV3 infection isat least 70% following administration of a single dose of the vaccine.

Further provided herein is a use of the vaccine of the presentdisclosure in the manufacture of a medicament for use in a method ofinducing an antigen specific immune response to hMPV/hPIV3 in a subject,the method comprising administering to the subject the vaccine in anamount effective to produce an antigen specific immune response tohMPV/hPIV3 in the subject.

Some embodiments provide a pharmaceutical composition for use invaccination of a subject comprising an effective dose of the vaccine ofthe present disclosure, wherein the effective dose is sufficient toproduce detectable levels of antigen as measured in serum of the subjectat 1-72 hours post administration. In some embodiments, the cut offindex of the antigen is 1-2. Some embodiments provide a pharmaceuticalcomposition for use in vaccination of a subject comprising an effectivedose of the vaccine of the present disclosure, wherein the effectivedose is sufficient to produce detectable levels of antigen as measuredin serum of the subject within 14 days hours post administration.

Other embodiments provide a pharmaceutical composition for use invaccination of a subject comprising an effective dose of the vaccine ofthe present disclosure, wherein the effective dose is sufficient toproduce a 1,000-10,000 neutralization titer produced by neutralizingantibody against hMPV/hPIV3 antigen as measured in serum of the subjectat 1-72 hours post administration. Yet other embodiments provide apharmaceutical composition for use in vaccination of a subjectcomprising an effective dose of the vaccine of the present disclosure,wherein the effective dose is sufficient to produce a 1,000-10,000neutralization titer produced by neutralizing antibody againsthMPV/hPIV3 antigen as measured in serum of the subject within 14 dayspost administration.

Further embodiments provide a vaccine, comprising (a) a humanmetapneumovirus (hMPV) ribonucleic acid (RNA) polynucleotide comprisingthe nucleic acid sequence identified by SEQ ID NO:4 or a nucleic acidsequence that is at least 95% identical to the nucleic acid sequenceidentified by SEQ ID NO:4, and (b) a human parainfluenza virus 3 (hPIV3)RNA polynucleotide comprising the nucleic acid sequence identified bySEQ ID NO:5 or a nucleic acid sequence that is at least 95% identical tothe nucleic acid sequence identified by SEQ ID NO:5, wherein the RNApolynucleotides of (a) and (b) are formulated in a lipid nanoparticlecomprising a cationic lipid, a PEG-modified lipid, a sterol and anon-cationic lipid.

Still further embodiments provide a vaccine, comprising (a) a humanmetapneumovirus (hMPV) ribonucleic acid (RNA) polynucleotide encoded bya nucleic acid comprising the nucleic acid sequence identified by SEQ IDNO:1 or a nucleic acid sequence that is at least 95% identical to thenucleic acid sequence identified by SEQ ID NO:1, and (b) a humanparainfluenza virus 3 (hPIV3) RNA polynucleotide encoded by a nucleicacid comprising the nucleic acid sequence identified by SEQ ID NO:2 or anucleic acid sequence that is at least 95% identical to the nucleic acidsequence identified by SEQ ID NO:2, wherein the RNA polynucleotides of(a) and (b) are formulated in a lipid nanoparticle comprising a cationiclipid, a PEG-modified lipid, a sterol and a non-cationic lipid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are graphs showing that cells transfected with (1) mRNAhaving an open reading frame (SEQ ID NO:4) encoding hMPV F protein (SEQID NO:7) and (2) mRNA having an open reading frame (SEQ ID NO:5)encoding hPIV3 F protein (SEQ ID NO:8) expressed hMPV protein and hPIV3protein on the cell surface. FIG. 1A, left panel, shows a hMPV Fprotein-positive (fluorescent) cell count for cells transfected with amock construct. FIG. 1A, right panel, shows a hMPV F protein-positive(fluorescent) cell count for cells transfected with hMPV/hPIV3 vaccineconstructs. hMPV F protein was detected using antibodies specific forhMPV F protein (MEP8). FIG. 1A, middle panel, shows fluorescenceobtained from untransfected control cells, using only a secondaryantibody. FIG. 1B shows the surface expression of hPIV3 F protein inHela cells detected using antibodies specific for hPIV3 F protein(MAB10207).

FIGS. 2A-2B are graphs showing the results of cotton rat hMPV and hPIV3challenge experiments using animals immunized with a vaccine containing(1) mRNA having an open reading frame (SEQ ID NO:4) encoding hMPV Fprotein (SEQ ID NO:7) and (2) mRNA having an open reading frame (SEQ IDNO:5) encoding hPIV3 F protein (SEQ ID NO:8) (see Table 2 for studydesign). FIG. 2A shows viral titers from the nose and lungs of cottonrats challenged with hMPV. FIG. 2B shows viral titers from the nose andlungs of cotton rats challenged with hPIV3. Cotton rats immunized withthe mRNA vaccine were protected from hMPV infection and hPIV3 infection.

FIGS. 3A-3B are graphs showing neutralizing antibody titers againsthMPV/A2 (FIG. 3A) or hPIV3 (FIG. 3B) from the serum of cotton ratsimmunized with a vaccine containing (1) mRNA having an open readingframe (SEQ ID NO:4) encoding hMPV F protein (SEQ ID NO:7) and (2) mRNAhaving an open reading frame (SEQ ID NO:5) encoding hPIV3 F protein (SEQID NO:8) formulated in either MC3 lipids or Compound 1 lipids (see Table2 for study design). The two formulations yielded comparable levels ofneutralizing antibody titers.

FIGS. 4A-4B are graphs showing hMPV (FIG. 4A) and PIV3 (FIG. 4B) serumneutralizing antibody titers in cotton rats. PIV3 neutralizing antibodytiters detected at high levels in all animals immunized with PIV3-Fand/or PIV3-HN mRNA (Groups 9-13).

FIGS. 5A-5B are graphs showing viral load after hMPV (FIG. 5A) or PIV3(FIG. 5B) challenge of cotton rats. High level of virus was detected inPBS control animals (Groups 5 and 14), but was close to or below thelimit of quantification in all mRNA-immunized animals (Groups 2-4 and9-13), demonstrating full protection in both the lung and the nose.

FIGS. 6A-6B are graphs showing the average lung pathology score afterhMPV (FIG. 6A) or PIV3 (FIG. 6B) challenge of cotton rats. AllmRNA-immunized animals (Groups 2-4 and 9-13) exhibited lunghistopathology scores equivalent to the PBS controls (Groups 5 and 14),indicating no vaccine-enhanced respiratory disease (ERD).

FIGS. 7A-7B are graphs showing results of African green monkey hMPV andhPIV3 challenge experiments using animals immunized with a vaccinecontaining (1) mRNA having an open reading frame (SEQ ID NO:4) encodinghMPV F protein (SEQ ID NO:7) and (2) mRNA having an open reading frame(SEQ ID NO:5) encoding hPIV3 F protein (SEQ ID NO:8) (see Table 3 forstudy design). FIG. 7A shows viral titers from the nose and lungs ofAfrican green monkeys challenged with hMPV. FIG. 7B shows viral titersfrom the nose and lungs of African green monkeys challenged with hPIV3.Sero-negative African green monkeys immunized with 2 doses of the mRNAvaccine were completely protected from hMPV infection and hPIV3infection.

FIGS. 8A-8D are graphs showing neutralizing antibody titers against hMPVin sero-negative African green monkeys immunized with two doses of avaccine containing (1) mRNA having an open reading frame (SEQ ID NO:4)encoding hMPV F protein (SEQ ID NO:7) and (2) mRNA having an openreading frame (SEQ ID NO:5) encoding hPIV3 F protein (SEQ ID NO:8) ondays 0 and 28 at 200 μg per dose (FIG. 8A), 100 μg per dose (FIG. 8B),10 μg per dose (FIG. 8C) per dose, or placebo (FIG. 8D). The mRNAvaccines were formulated with Compound 1 lipids.

FIGS. 9A-9C are graphs showing neutralizing antibody titers against hMPVin sero-negative African green monkeys immunized with one dose of avaccine containing (1) mRNA having an open reading frame (SEQ ID NO:4)encoding hMPV F protein (SEQ ID NO:7) and (2) mRNA having an openreading frame (SEQ ID NO:5) encoding hPIV3 F protein (SEQ ID NO:8) onday 0 at 200 μg per dose (FIG. 9A), 100 μg per dose (FIG. 9B), or 50 μgper dose (FIG. 9C) per dose. The mRNA vaccines were formulated withCompound 1 lipids.

FIGS. 10A-10D are graphs showing the neutralizing antibody titersagainst hPIV3 in sero-negative African green monkeys immunized with twodoses of a vaccine containing (1) mRNA having an open reading frame (SEQID NO:4) encoding hMPV F protein (SEQ ID NO:7) and (2) mRNA having anopen reading frame (SEQ ID NO:5) encoding hPIV3 F protein (SEQ ID NO:8)on days 0 and 28 at 200 μg per dose (FIG. 10A), 100 μg per dose (FIG.10B), 10 μg per dose (FIG. 10C) per dose, or placebo (FIG. 10D). ThemRNA vaccines were formulated with Compound 1 lipids.

FIGS. 11A-11B are graphs showing the neutralizing antibody titersagainst hPIV3 in sero-negative African green monkeys immunized with two200 μg doses a vaccine containing mRNA having an open reading frame (SEQID NO:5) encoding hPIV3 F protein (SEQ ID NO:8) (FIG. 11A) or a vaccinecontaining mRNA having an open reading frame (SEQ ID NO:6) encodinghPIV3 HN protein (SEQ ID NO:9) (FIG. 11B). The vaccine encoding hPIV3-Fprotein induced higher neutralizing antibody titers than the vaccineencoding hPIV3-HN. The mRNA vaccines were formulated with Compound 1lipids.

FIGS. 12A-12C are graphs showing the neutralizing antibody titersagainst hPIV3 in sero-negative African green monkeys immunized with onedose of a vaccine containing (1) mRNA having an open reading frame (SEQID NO:4) encoding hMPV F protein (SEQ ID NO:7) and (2) mRNA having anopen reading frame (SEQ ID NO:5) encoding hPIV3 F protein (SEQ ID NO:8)on days 0 at 200 μg per dose (FIG. 12B), 50 μg per dose (FIG. 12C), orplacebo (FIG. 12A). The mRNA vaccines were formulated with Compound 1lipids.

FIGS. 13A-13B are graphs showing the neutralizing antibodies againsthMPV/A2 in sero-negative African green monkeys or sero-positive Africangreen monkeys. FIG. 13A shows that 2 doses of 200 μg and 100 μg of avaccine containing (1) mRNA having an open reading frame (SEQ ID NO:4)encoding hMPV F protein (SEQ ID NO:7) and (2) mRNA having an openreading frame (SEQ ID NO:5) encoding hPIV3 F protein (SEQ ID NO:8)elicits high levels of hMPV neutralizing antibodies. FIG. 13B shows thata single dose of the hMPVhPIV3 mRNA vaccine boosts hMPV neutralizingantibodies by 8-10 fold in sero-positive African green monkeys.

FIGS. 14A-14B are graphs showing the neutralizing antibodies againsthPIV3 in sero-negative African green monkeys (AGMs) or sero-positiveAfrican green monkeys. FIG. 14A shows that 2 doses of 200 μg and 100 μgof a vaccine containing (1) mRNA having an open reading frame (SEQ IDNO:4) encoding hMPV F protein (SEQ ID NO:7) and (2) mRNA having an openreading frame (SEQ ID NO:5) encoding hPIV3 F protein (SEQ ID NO:8)elicits high levels of hMPV neutralizing antibodies. FIG. 14B shows thata single dose of the hMPV/hPIV3 mRNA vaccine boosts hPIV3 neutralizingantibodies by 4-10 folds in sero-positive African green monkeys.

FIGS. 15A-15B are graphs showing the neutralizing antibody titers tohMPV (FIG. 15A) and hPIV3 (FIG. 15B). hMPV or PIV3 neutralizing antibodytiters could be detected in all previously-exposed AGM (Groups 11-13 forhMPV and Groups 14-16 for PIV3) and were stable for the 4 weekspreceding immunization. In all cases the peak neutralizing antibodyresponse was reached by 14 days post immunization, and was generallystable for the subsequent 42 days.

FIGS. 16A-16B are graphs showing the neutralizing antibody titers tohMPV (FIG. 16A) and hPIV3 (FIG. 16B) in AGM. hMPV neutralizingantibodies were detected in serum of the majority of animals 28 daysafter the first immunization with the hMPV-F/PIV3-F/PIV3-HN mRNAvaccine, and titers were boosted by the second immunization. PIV3neutralizing antibodies were detected in serum of the majority ofanimals 28 days after the first immunization with the hMPV/PIV3 mRNAvaccines, and titers were boosted by the second immunization.

FIGS. 17A-17B are graphs showing viral load after hMPV (FIG. 17A) orhPIV3 (FIG. 17B) challenge of AGM. The hMPV/PIV3 combination vaccineaffords full protection against both viruses in the lung and the nose.

DETAILED DESCRIPTION

The present disclosure provides, in some embodiments, combinationvaccine therapies that comprise administering RNA (e.g., mRNA)polynucleotides encoding a human metapneumovirus (hMPV) F protein and ahuman parainfluenza virus type 3 (hPIV3) F protein, either formulated asa combination vaccine or formulated as single vaccines administeredsimultaneously or sequentially. In some embodiments, a combinationvaccine may further comprise a RNA (e.g., mRNA) polynucleotide encodinga respiratory syncytial virus (RSV) antigenic polypeptide.

Also provided herein are vaccines (vaccine compositions) comprising RNAencoding hMPV F protein and/or hPIV3 F protein, methods of manufacturingthese vaccines, and nucleic acids encoding these vaccines.

For simplicity, the term “hMPV/hPIV3” should be understood to encompasshMPV, hPIV3, or both hMPV and hPIV3. “hMPV/hPIV3” compositions contain,for example, a mRNA encoding hMPV, a mRNA encoding hPIV3 as well as oneor more additional mRNAs encoding respiratory antigens (e.g., RSVantigens).

The hMPV/hPIV3 RNA (e.g., mRNA) vaccines, in some embodiments, areformulated in a lipid nanoparticle comprising a cationic lipid, aPEG-modified lipid, a sterol and a non-cationic lipid. In someembodiments, the non-cationic lipid is Compound 1 of Formula (I). Thus,in some embodiments, a hMPV/PIV3 RNA vaccine is formulated in a lipidnanoparticle that comprises Compound 1.

In some embodiments, the hMPV/hPIV3 RNA (e.g., mRNA) vaccines may beused to treat a lower respiratory hMPV and/or hPIV3 infection in achild, an elderly person, a young adult, or an immunocompromised person.The hMPV/hPIV3 RNA (e.g., mRNA) vaccines, in some embodiments, may beused to induce a balanced immune response, comprising both cellular andhumoral immunity, without many of the risks associated with DNAvaccination. It has been discovered that the mRNA vaccines describedherein are superior to current vaccines in several ways. First, thelipid nanoparticle (LNP) delivery is superior to other formulationsincluding a protamine base approach described in the literature and noadditional adjuvants are to be necessary. The use of LNPs enables theeffective delivery of chemically modified or unmodified mRNA vaccines.Additionally it has been demonstrated herein that both modified andunmodified LNP formulated mRNA vaccines were superior to conventionalvaccines by a significant degree. In some embodiments the mRNA vaccinesof the present disclosure are superior to conventional vaccines by afactor of at least 10 fold, 20 fold, 40 fold, 50 fold, 100 fold, 500fold or 1,000 fold.

In addition, the vaccines of the present disclosure result in effectiveimmune responses against both hMPV and PIV3 (e.g., as measured by areduction in infectious virus isolated from the nasal and/or lungpassages upon exposure to virus), but do not result in visiblerespiratory pathology (e.g., alveolitis (cells within the alveolarspaces) or interstitial pneumonia (inflammatory cell infiltration andthickening of alveolar walls)). For example, viral load (e.g., asdetermined by plaque assay and pulmonary histopathology) was evaluatedon hematoxylin and eosin (H&E) stained fixed lung sections of vaccinatedanimals. The sections were evaluated on a 0-4 severity scale andsubsequently converted to a 0-100% histopathology scale. The lunghistopathology scores were equivalent to the control, indicating novaccine-enhanced respiratory disease (ERD).

Although attempts have been made to produce functional RNA vaccines,including mRNA vaccines and self-replicating RNA vaccines, thetherapeutic efficacy of these RNA vaccines have not yet been fullyestablished. Quite surprisingly, the inventors have discovered,according to aspects of the present disclosure a class of formulationsfor delivering mRNA vaccines in vivo that results in significantlyenhanced, and in many respects synergistic, immune responses includingenhanced antigen generation and functional antibody production withneutralization capability. These results can be achieved even whensignificantly lower doses of the mRNA are administered in comparisonwith mRNA doses used in other classes of lipid based formulations. Theformulations of the present disclosure have demonstrated significantunexpected in vivo immune responses sufficient to establish the efficacyof functional mRNA vaccines as prophylactic and therapeutic agents.Additionally, self-replicating RNA vaccines rely on viral replicationpathways to deliver enough RNA to a cell to produce an immunogenicresponse. The formulations of the present disclosure do not requireviral replication to produce enough protein to result in a strong immuneresponse. Thus, the mRNA of the present disclosure are notself-replicating RNA and do not include components necessary for viralreplication.

The present disclosure involves, in some aspects, the surprising findingthat lipid nanoparticle (LNP) formulations significantly enhance theeffectiveness of mRNA vaccines, including chemically modified andunmodified mRNA vaccines. The efficacy of mRNA vaccines formulated inLNP was examined in vivo using several distinct antigens. The resultspresented herein demonstrate the unexpected superior efficacy of themRNA vaccines formulated in LNP over other commercially availablevaccines.

In addition to providing an enhanced immune response, the formulationsof the present disclosure generate a more rapid immune response withfewer doses of antigen than other vaccines tested. The mRNA-LNPformulations of the present disclosure also produce quantitatively andqualitatively better immune responses than vaccines formulated in adifferent carriers.

The LNP used in the studies described herein has been used previously todeliver siRNA in various animal models as well as in humans. In view ofthe observations made in association with the siRNA delivery of LNPformulations, the fact that LNP is useful in vaccines is quitesurprising. It has been observed that therapeutic delivery of siRNAformulated in LNP causes an undesirable inflammatory response associatedwith a transient IgM response, typically leading to a reduction inantigen production and a compromised immune response. In contrast to thefindings observed with siRNA, the LNP-mRNA formulations of the presentdisclosure are demonstrated herein to generate enhanced IgG levels,sufficient for prophylactic and therapeutic methods rather thantransient IgM responses.

hHMPV/hPIV3 RNA Vaccine Compositions

In some embodiments, a vaccine of the present disclosure comprises a RNA(e.g., mRNA) polynucleotide encoding a human metapneumovirus (hMPV)fusion (F) protein. In other embodiments, a vaccine of the presentdisclosure comprises a RNA (e.g., mRNA) polynucleotide encoding a humanparainfluenza virus type 3 (hPIV3) fusion (F) protein. In yet otherembodiments, a vaccine of the present disclosure comprises a RNA (e.g.,mRNA) polynucleotide encoding a hMPV F protein and a RNA (e.g., mRNA)polynucleotide encoding a hPIV3 F protein.

In some embodiments, the vaccine of the present disclosure comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV F protein comprising theamino acid sequence identified by SEQ ID NO:7. In some embodiments, avaccine of the present disclosure comprises a RNA (e.g., mRNA)polynucleotide encoding a hMPV F protein comprising an amino acidsequence that is at least 85% identical to the amino acid sequenceidentified by SEQ ID NO:7. For example, a vaccine may comprises a RNA(e.g., mRNA) polynucleotide encoding a hMPV F protein comprising anamino acid sequence that is at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to the amino acid sequenceidentified by SEQ ID NO:7. In some embodiments, a vaccine comprises aRNA (e.g., mRNA) polynucleotide encoding a hMPV F protein comprising anamino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequenceidentified by SEQ ID NO:7.

In some embodiments, a vaccine of the present disclosure comprises a RNA(e.g., mRNA) polynucleotide comprising the nucleotide sequenceidentified by SEQ ID NO:4. In some embodiments, a vaccine comprises aRNA (e.g., mRNA) polynucleotide comprising a nucleotide sequence that isat least 85% identical to the nucleotide sequence identified by SEQ IDNO:4. For example, a vaccine may comprise a RNA (e.g., mRNA)polynucleotide comprising a nucleotide sequence that is at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to thenucleotide sequence identified by SEQ ID NO:4. In some embodiments, avaccine of the present disclosure comprises a RNA (e.g., mRNA)polynucleotide comprising a nucleotide sequence that is 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto the nucleotide sequence identified by SEQ ID NO:4.

In some embodiments, a vaccine of the present disclosure comprises a RNA(e.g., mRNA) polynucleotide encoding a hPIV3 F protein comprising theamino acid sequence identified by SEQ ID NO:8. In some embodiments, avaccine comprises a RNA (e.g., mRNA) polynucleotide encoding a hPIV3 Fprotein comprising an amino acid sequence that is at least 85% identicalto the amino acid sequence identified by SEQ ID NO:8. For example, avaccine may comprise a RNA (e.g., mRNA) polynucleotide encoding a hPIV3F protein comprising an amino acid sequence that is at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to theamino acid sequence identified by SEQ ID NO:8. In some embodiments, avaccine comprises a RNA (e.g., mRNA) polynucleotide encoding a hPIV3 Fprotein comprising an amino acid sequence that is 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical tothe amino acid sequence identified by SEQ ID NO:8.

In some embodiments, a vaccine of the present disclosure comprises a RNA(e.g., mRNA) polynucleotide comprising a nucleotide sequence identifiedby SEQ ID NO:5. In some embodiments, a vaccine comprises a RNA (e.g.,mRNA) polynucleotide comprising a nucleotide sequence that is at least85% identical to the nucleotide sequence identified by SEQ ID NO:5. Forexample, a vaccine may comprise a RNA (e.g., mRNA) polynucleotidecomprising a nucleotide sequence that is at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to the nucleotidesequence identified by SEQ ID NO:5. In some embodiments, a vaccinecomprises a RNA (e.g., mRNA) polynucleotide comprising a nucleotidesequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the nucleotide sequence identified bySEQ ID NO:5.

In some embodiments, the RNA (e.g., mRNA) vaccine of the presentdisclosure comprises a RNA (e.g., mRNA) polynucleotide encoding a hPIV3hemagglutinin-neuraminidase (UN) comprising the amino acid sequenceidentified by SEQ ID NO:9. In some embodiments, the RNA (e.g., mRNA)vaccine of the present disclosure comprises a RNA (e.g., mRNA)polynucleotide encoding a hPIV3 hemagglutinin-neuraminidase (HN)comprising an amino acid sequence that is at least 85% identical to theamino acid sequence identified by SEQ ID NO:9. For example, the RNA(e.g., mRNA) vaccine of the present disclosure comprises a RNA (e.g.,mRNA) polynucleotide encoding a hPIV3 hemagglutinin-neuraminidase (HN)comprising an amino acid sequence that is at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to the amino acidsequence identified by SEQ ID NO:9. In some embodiments, the RNA (e.g.,mRNA) vaccine of the present disclosure comprises a RNA (e.g., mRNA)polynucleotide encoding a hPIV3 hemagglutinin-neuraminidase (HN)comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aminoacid sequence identified by SEQ ID NO:9.

In some embodiments, a vaccine of the present disclosure comprises a RNA(e.g., mRNA) polynucleotide comprising a nucleotide sequence identifiedby SEQ ID NO:6. In some embodiments, a vaccine comprises a RNA (e.g.,mRNA) polynucleotide comprising a nucleotide sequence that is at least85% identical to the nucleotide sequence identified by SEQ ID NO:6. Forexample, a vaccine may comprise a RNA (e.g., mRNA) polynucleotidecomprising a nucleotide sequence that is at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to the nucleotidesequence identified by SEQ ID NO:6. In some embodiments, a vaccinecomprises a RNA (e.g., mRNA) polynucleotide comprising a nucleotidesequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to the nucleotide sequence identified bySEQ ID NO:6.

In some embodiments, a vaccine of the present disclosure is acombination vaccine comprising a RNA (e.g., mRNA) polynucleotideencoding a hMPV F protein and a RNA (e.g., mRNA) polynucleotide encodinga hPIV3 F protein. In some embodiments, a vaccine comprises (a) a RNA(e.g., mRNA) polynucleotide encoding a hMPV F protein comprising theamino acid sequence identified by SEQ ID NO:7 or an amino acid sequencethat is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99%) identical to the amino acid sequence identified by SEQ ID NO:7,and (b) a RNA polynucleotide encoding a hPIV3 F protein comprising theamino acid sequence identified by SEQ ID NO:8 or an amino acid sequencethat is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99%) identical to the amino acid sequence identified by SEQ ID NO:8.

In some embodiments, a vaccine of the present disclosure is acombination vaccine comprising (a) a hMPV ribonucleic acid (RNA)polynucleotide comprising the nucleic acid sequence identified by SEQ IDNO:4 or a nucleic acid sequence that is at least 90% (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acidsequence identified by SEQ ID NO:4, and (b) a hPIV3 RNA polynucleotidecomprising the nucleic acid sequence identified by SEQ ID NO:5 or anucleic acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid sequenceidentified by SEQ ID NO:5.

In some embodiments, a vaccine of the present disclosure is acombination vaccine comprising (a) a hMPV ribonucleic acid (RNA)polynucleotide encoded by a nucleic acid comprising the nucleic acidsequence identified by SEQ ID NO:1 or a nucleic acid sequence that is atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%)identical to the nucleic acid sequence identified by SEQ ID NO:1, and(b) a hPIV3 RNA polynucleotide encoded by a nucleic acid comprising thenucleic acid sequence identified by SEQ ID NO:2 or a nucleic acidsequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%) identical to the nucleic acid sequence identified bySEQ ID NO:2.

hMPV/hPIV3 Fusion (F) Proteins and Other Antigens/Antigenic Polypeptides

Human Metapneumovirus (hMPV) F Protein. hMPV shares substantial homologywith respiratory syncytial virus (RSV) in its surface glycoproteins.hMPV fusion protein (F) is related to other paramyxovirus fusionproteins and appears to have homologous regions that may have similarfunctions. The hMPV F protein amino acid sequence contains featurescharacteristic of other paramyxovirus F proteins, including a putativecleavage site and potential N-linked glycosylation sites. ParamyxovirusF proteins are synthesized as inactive precursors (FO) that are cleavedby host cell proteases into the biologically fusion-active F1 and F2domains (see, e.g., Cseke G. et al. Journal of Virology 2007;81(2):698-707, incorporated herein by reference). hMPV has one putativecleavage site, in contrast to the two sites established for RSV F, andonly shares 34% amino acid sequence identity with RSV F. F2 isextracellular and disulfide linked to F1. F proteins are type Iglycoproteins existing as trimers, with two 4-3 heptad repeat domains atthe N- and C-terminal regions of the protein (HR1 and HR2), which formcoiled-coil alpha-helices. These coiled coils become apposed in anantiparallel fashion when the protein undergoes a conformational changeinto the fusogenic state. There is a hydrophobic fusion peptide Nproximal to the N-terminal heptad repeat, which is thought to insertinto the target cell membrane, while the association of the heptadrepeats brings the transmembrane domain into close proximity, inducingmembrane fusion (see, e.g., Baker, K A et al. Mol. Cell 1999;3:309-319). This mechanism has been proposed for a number of differentviruses, including RSV, influenza virus, and human immunodeficiencyvirus. F proteins are major antigenic determinants for all knownparamyxoviruses and for other viruses that possess similar fusionproteins such as human immunodeficiency virus, influenza virus, andEbola virus.

Human Parainfluenza Virus Type 3 (hPIV3) F Protein. Parainfluenzaviruses belong to the family Paramyxoviridae. These are envelopedviruses with a negative-sense single-stranded RNA genome. Parainfluenzaviruses belong to the subfamily Paramyxoviridae, which is subdividedinto three genera: Respirovirus (PIV-1, PIV-3, and Sendai virus (SeV)),Rubulavirus (PIV-2, PIV-4 and mumps virus) and Morbillivirus (measlesvirus, rinderpest virus and canine distemper virus (CDV)). Their genome,a ˜15,500 nucleotide-long negative-sense RNA molecule, encodes twoenvelope glycoproteins, the hemagglutinin-neuraminidase (HN), the fusionprotein (F or F0), which is cleaved into F1 and F2 subunits, a matrixprotein (M), a nucleocapsid protein (N) and several nonstructuralproteins including the viral replicase (L). All parainfluenza viruses,except for PIV1, express a non-structural V protein that blocks IFNsignaling in the infected cell and acts therefore as a virulence factor(see, e.g., Nishio M et al. J Virol. 2008; 82(13):6130-38).

PIV3 fusion protein (PIV3 F) is located on the viral envelope, where itfacilitates the viral fusion and cell entry. The F protein is initiallyinactive, but proteolytic cleavage leads to its active forms, F1 and F2,which are linked by disulfide bonds. This occurs when the HN proteinbinds its receptor on the host cell's surface. During early phases ofinfection, the F glycoprotein mediates penetration of the host cell byfusion of the viral envelope to the plasma membrane. In later stages ofthe infection, the F protein facilitates the fusion of the infectedcells with neighboring uninfected cells, which leads to the formation ofa syncytium and spread of the infection.

An antigenic polypeptide (e.g., hMPV/hPIV3 F protein) encoded by a RNAvaccine of the present disclosure may be naturally occurringpolypeptides, synthetic polypeptides, homologs, orthologs, paralogs,fragments and other equivalents, variants, and analogs of the foregoing.A polypeptide may be a single molecule or may be a multi-molecularcomplex such as a dimer, trimer or tetramer. Polypeptides may alsocomprise single chain polypeptides or multichain polypeptides, such asantibodies or insulin, and may be associated or linked to each other.Most commonly, disulfide linkages are found in multichain polypeptides.The term “polypeptide” may also apply to amino acid polymers in which atleast one amino acid residue is an artificial chemical analogue of acorresponding naturally-occurring amino acid.

A “polypeptide variant” is a molecule that differs in its amino acidsequence relative to a native sequence or a reference sequence. Aminoacid sequence variants may possess substitutions, deletions, insertions,or a combination of any two or three of the foregoing, at certainpositions within the amino acid sequence, as compared to a nativesequence or a reference sequence. Ordinarily, variants possess at least50% identity to a native sequence or a reference sequence. In someembodiments, variants share at least 80% identity or at least 90%identity with a native sequence or a reference sequence.

Vaccines of the present disclosure may include a variant of a hMPVand/or hPIV3 F protein. These include, for example, substitutional,insertional, deletion and covalent variants and derivatives. The term“derivative” is synonymous with the term “variant” and generally refersto a molecule that has been modified and/or changed in any way relativeto a reference molecule or a starting molecule.

As such, polynucleotides encoding peptides or polypeptides containingsubstitutions, insertions and/or additions, deletions and covalentmodifications with respect to reference sequences, in particular thepolypeptide sequences disclosed herein, are included within the scope ofthis disclosure. For example, sequence tags or amino acids, such as oneor more lysines, can be added to peptide sequences (e.g., at theN-terminal or C-terminal ends). Sequence tags can be used for peptidedetection, purification or localization. Lysines can be used to increasepeptide solubility or to allow for biotinylation. Alternatively, aminoacid residues located at the carboxy and amino terminal regions of theamino acid sequence of a peptide or protein may optionally be deletedproviding for truncated sequences. Certain amino acids (e.g., C-terminalresidues or N-terminal residues) alternatively may be deleted dependingon the use of the sequence, as for example, expression of the sequenceas part of a larger sequence that is soluble, or linked to a solidsupport.

“Substitutional variants” when referring to polypeptides are those thathave at least one amino acid residue in a native or starting sequenceremoved and a different amino acid inserted in its place at the sameposition. Substitutions may be single, where only one amino acid in themolecule has been substituted, or they may be multiple, where two ormore (e.g., 3, 4 or 5) amino acids have been substituted in the samemolecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Features” when referring to polypeptide or polynucleotide are definedas distinct amino acid sequence-based or nucleotide-based components ofa molecule respectively. Features of the polypeptides encoded by thepolynucleotides include surface manifestations, local conformationalshape, folds, loops, half-loops, domains, half-domains, sites, terminiand any combination(s) thereof.

When referring to polypeptides the term “domain” refers to a motif of apolypeptide having one or more identifiable structural or functionalcharacteristics or properties (e.g., binding capacity, serving as a sitefor protein-protein interactions).

When referring to polypeptides the terms “site” as it pertains to aminoacid based embodiments is used synonymously with “amino acid residue”and “amino acid side chain.” As used herein when referring topolynucleotides the terms “site” as it pertains to nucleotide basedembodiments is used synonymously with “nucleotide.” A site represents aposition within a peptide or polypeptide or polynucleotide that may bemodified, manipulated, altered, derivatized or varied within thepolypeptide-based or polynucleotide-based molecules.

The terms “termini” or “terminus” when referring to polypeptides orpolynucleotides refers to an extremity of a polypeptide orpolynucleotide respectively. Such extremity is not limited only to thefirst or final site of the polypeptide or polynucleotide but may includeadditional amino acids or nucleotides in the terminal regions.Polypeptide-based molecules may be characterized as having both anN-terminus (terminated by an amino acid with a free amino group (NH2))and a C-terminus (terminated by an amino acid with a free carboxyl group(COOH)). Proteins are in some cases made up of multiple polypeptidechains brought together by disulfide bonds or by non-covalent forces(multimers, oligomers). These proteins have multiple N- and C-termini.Alternatively, the termini of the polypeptides may be modified such thatthey begin or end, as the case may be, with a non-polypeptide basedmoiety such as an organic conjugate.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of polypeptides of interest. For example, providedherein is any protein fragment (meaning a polypeptide sequence at leastone amino acid residue shorter than a reference polypeptide sequence butotherwise identical) of a reference protein having a length of 10, 20,30, 40, 50, 60, 70, 80, 90, 100 or longer than 100 amino acids. Inanother example, any protein that includes a stretch of 20, 30, 40, 50,or 100 (contiguous) amino acids that are 40%, 50%, 60%, 70%, 80%, 90%,95%, or 100% identical to any of the sequences described herein can beutilized in accordance with the disclosure. In some embodiments, apolypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations asshown in any of the sequences provided herein or referenced herein. Inanother example, any protein that includes a stretch of 20, 30, 40, 50,or 100 amino acids that are greater than 80%, 90%, 95%, or 100%identical to any of the sequences described herein, wherein the proteinhas a stretch of 5, 10, 15, 20, 25, or 30 amino acids that are less than80%, 75%, 70%, 65% to 60% identical to any of the sequences describedherein can be utilized in accordance with the disclosure.

Polypeptide or polynucleotide molecules of the present disclosure mayshare a certain degree of sequence identity with the reference molecules(e.g., reference polypeptides or reference polynucleotides), forexample, an F protein having an amino acid sequence identified by SEQ IDNO:7 or SEQ ID NO:8. The term “identity” refers to the overallrelatedness between polymeric molecules, for example, betweenpolynucleotide molecules (e.g. DNA molecules and/or RNA molecules)and/or between polypeptide molecules. Calculation of the percentidentity of two polynucleic acid sequences, for example, can beperformed by aligning the two sequences for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondnucleic acid sequences for optimal alignment and non-identical sequencescan be disregarded for comparison purposes). In certain embodiments, thelength of a sequence aligned for comparison purposes is at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or 100% of the length of the referencesequence. The nucleotides at corresponding nucleotide positions are thencompared. When a position in the first sequence is occupied by the samenucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position. The percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences, taking into account the number ofgaps, and the length of each gap, which needs to be introduced foroptimal alignment of the two sequences. The comparison of sequences anddetermination of percent identity between two sequences can beaccomplished using a mathematical algorithm. For example, the percentidentity between two nucleic acid sequences can be determined usingmethods such as those described in Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Sequence Analysis in Molecular Biology, von Heinje, G.,Academic Press, 1987; Computer Analysis of Sequence Data, Part I,Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds.,M Stockton Press, New York, 1991; each of which is incorporated hereinby reference. For example, the percent identity between two nucleic acidsequences can be determined using the algorithm of Meyers and Miller(CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGNprogram (version 2.0) using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. The percent identity between twonucleic acid sequences can, alternatively, be determined using the GAPprogram in the GCG software package using an NWSgapdna.CMP matrix.Methods commonly employed to determine percent identity betweensequences include, but are not limited to those disclosed in Carillo,H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporatedherein by reference. Techniques for determining identity are codified inpublicly available computer programs. Exemplary computer software todetermine homology between two sequences include, but are not limitedto, GCG program package, Devereux, J., et al., Nucleic Acids Research,12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J.Molec. Biol., 215, 403 (1990)).

Thus, the term “identity” refers to a relationship between the sequencesof two or more polypeptides or polynucleotides, as determined bycomparing the sequences. In the art, identity also means the degree ofsequence relatedness between two sequences as determined by the numberof matches between strings of two or more amino acid residues or nucleicacid residues. Identity measures the percent of identical matchesbetween the smaller of two or more sequences with gap alignments (ifany) addressed by a particular mathematical model or computer program(e.g., “algorithms”). Identity of related peptides can be readilycalculated by known methods. “% identity” as it applies to polypeptideor polynucleotide sequences is defined as the percentage of residues(amino acid residues or nucleic acid residues) in the candidate aminoacid or nucleic acid sequence that are identical with the residues inthe amino acid sequence or nucleic acid sequence of a second sequenceafter aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent identity. Methods and computer programs forthe alignment are well known in the art. Identity depends on acalculation of percent identity but may differ in value due to gaps andpenalties introduced in the calculation. Generally, variants of aparticular polynucleotide or polypeptide have at least 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% but less than 100% sequence identity to that particularreference polynucleotide or polypeptide as determined by sequencealignment programs and parameters described herein and known to thoseskilled in the art. Such tools for alignment include those of the BLASTsuite (Stephen F. Altschul, et al. (1997).” Gapped BLAST and PSI-BLAST:a new generation of protein database search programs,” Nucleic AcidsRes. 25:3389-3402). Another popular local alignment technique is basedon the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981)“Identification of common molecular subsequences.” J. Mol. Biol.147:195-197). A general global alignment technique based on dynamicprogramming is the Needleman-Wunsch algorithm (Needleman, S. B. &Wunsch, C. D. (1970) “A general method applicable to the search forsimilarities in the amino acid sequences of two proteins.” J. Mol. Biol.48:443-453). More recently, a Fast Optimal Global Sequence AlignmentAlgorithm (FOGSAA) was developed that purportedly produces globalalignment of nucleotide and protein sequences faster than other optimalglobal alignment methods, including the Needleman-Wunsch algorithm.

The term “homology” refers to the overall relatedness between polymericmolecules, e.g. between nucleic acid molecules (e.g. DNA moleculesand/or RNA molecules) and/or between polypeptide molecules. Polymericmolecules (e.g. nucleic acid molecules (e.g. DNA molecules and/or RNAmolecules) and/or polypeptide molecules) that share a threshold level ofsimilarity or identity determined by alignment of matching residues aretermed homologous. Homology is a qualitative term that describes arelationship between molecules and can be based upon the quantitativesimilarity or identity. Similarity or identity is a quantitative termthat defines the degree of sequence match between two comparedsequences. In some embodiments, polymeric molecules are considered to be“homologous” to one another if their sequences are at least 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%identical or similar. The term “homologous” necessarily refers to acomparison between at least two sequences (polynucleotide or polypeptidesequences). Two polynucleotide sequences are considered homologous ifthe polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%,or even 99% for at least one stretch of at least 20 amino acids. In someembodiments, homologous polynucleotide sequences are characterized bythe ability to encode a stretch of at least 4-5 uniquely specified aminoacids. For polynucleotide sequences less than 60 nucleotides in length,homology is determined by the ability to encode a stretch of at least4-5 uniquely specified amino acids. Two protein sequences are consideredhomologous if the proteins are at least 50%, 60%, 70%, 80%, or 90%identical for at least one stretch of at least 20 amino acids.

Homology implies that the compared sequences diverged in evolution froma common origin. The term “homolog” refers to a first amino acidsequence or nucleic acid sequence (e.g., gene (DNA or RNA) or proteinsequence) that is related to a second amino acid sequence or nucleicacid sequence by descent from a common ancestral sequence. The term“homolog” may apply to the relationship between genes and/or proteinsseparated by the event of speciation or to the relationship betweengenes and/or proteins separated by the event of genetic duplication.“Orthologs” are genes (or proteins) in different species that evolvedfrom a common ancestral gene (or protein) by speciation. Typically,orthologs retain the same function in the course of evolution.“Paralogs” are genes (or proteins) related by duplication within agenome. Orthologs retain the same function in the course of evolution,whereas paralogs evolve new functions, even if these are related to theoriginal one.

Nucleic Acids/Polynucleotides

The term “nucleic acid” includes any compound and/or substance thatcomprises a polymer of nucleotides (nucleotide monomer). These polymersare referred to as polynucleotides. Thus, the terms “nucleic acid” and“polynucleotide” are used interchangeably.

Nucleic acids may be or may include, for example, ribonucleic acids(RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs),glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), lockednucleic acids (LNAs, including LNA having a β-D-ribo configuration,α-LNA having an α-L-ribo configuration (a diastereomer of LNA),2′-amino-LNA having a 2′-amino functionalization, and 2′-amino-α-LNAhaving a 2′-amino functionalization), ethylene nucleic acids (ENA),cyclohexenyl nucleic acids (CeNA) or chimeras or combinations thereof.

In some embodiments, polynucleotides of the present disclosure functionas messenger RNA (mRNA). “Messenger RNA” (mRNA) refers to anypolynucleotide that encodes a (at least one) polypeptide (anaturally-occurring, non-naturally-occurring, or modified polymer ofamino acids) and can be translated to produce the encoded polypeptide invitro, in vivo, in situ or ex vivo. The skilled artisan will appreciatethat, except where otherwise noted, polynucleotide sequences set forthin the instant application will recite “T”s in a representative DNAsequence but where the sequence represents RNA (e.g., mRNA), the “T”swould be substituted for “U”s. Thus, any of the RNA polynucleotidesencoded by a DNA identified by a particular sequence identificationnumber may also comprise the corresponding RNA (e.g., mRNA) sequenceencoded by the DNA, where each “T” of the DNA sequence is substitutedwith “U.”

The basic components of an mRNA molecule typically include at least onecoding region, a 5′ untranslated region (UTR), a 3′ UTR, a 5′ cap and apoly-A tail. Polynucleotides of the present disclosure may function asmRNA but can be distinguished from wild-type mRNA in their functionaland/or structural design features, which serve to overcome existingproblems of effective polypeptide expression using nucleic-acid basedtherapeutics.

Polynucleotides of the present disclosure, in some embodiments, arecodon optimized. Codon optimization methods are known in the art and maybe used as provided herein. Codon optimization, in some embodiments, maybe used to match codon frequencies in target and host organisms toensure proper folding; bias GC content to increase mRNA stability orreduce secondary structures; minimize tandem repeat codons or base runsthat may impair gene construction or expression; customizetranscriptional and translational control regions; insert or removeprotein trafficking sequences; remove/add post translation modificationsites in encoded protein (e.g. glycosylation sites); add, remove orshuffle protein domains; insert or delete restriction sites; modifyribosome binding sites and mRNA degradation sites; adjust translationalrates to allow the various domains of the protein to fold properly; orto reduce or eliminate problem secondary structures within thepolynucleotide. Codon optimization tools, algorithms and services areknown in the art—non-limiting examples include services from GeneArt(Life Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietarymethods. In some embodiments, the open reading frame (ORF) sequence isoptimized using optimization algorithms.

In some embodiments, a codon optimized sequence shares less than 95%sequence identity, less than 90% sequence identity, less than 85%sequence identity, less than 80% sequence identity, or less than 75%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., F protein)).

In some embodiments, a codon-optimized sequence shares between 65% and85% (e.g., between about 67% and about 85%, or between about 67% andabout 80%) sequence identity to a naturally-occurring sequence or awild-type sequence (e.g., a naturally-occurring or wild-type mRNAsequence encoding a polypeptide or protein of interest (e.g., anantigenic protein or polypeptide)). In some embodiments, acodon-optimized sequence shares between 65% and 75%, or about 80%sequence identity to a naturally-occurring sequence or wild-typesequence (e.g., a naturally-occurring or wild-type mRNA sequenceencoding a polypeptide or protein of interest (e.g., an antigenicprotein or polypeptide)).

In some embodiments a codon-optimized RNA (e.g., mRNA) may, forinstance, be one in which the levels of G/C are enhanced. TheG/C-content of nucleic acid molecules may influence the stability of theRNA. RNA having an increased amount of guanine (G) and/or cytosine (C)residues may be functionally more stable than nucleic acids containing alarge amount of adenine (A) and thymine (T) or uracil (U) nucleotides.WO02/098443 discloses a pharmaceutical composition containing an mRNAstabilized by sequence modifications in the translated region. Due tothe degeneracy of the genetic code, the modifications work bysubstituting existing codons for those that promote greater RNAstability without changing the resulting amino acid. The approach islimited to coding regions of the RNA.

Variants

In some embodiments, an RNA of the present disclosure encodes ahMPV/hPIV3 antigen variant. Antigen or other polypeptide variants refersto molecules that differ in their amino acid sequence from a wild-type,native or reference sequence. The antigen/polypeptide variants maypossess substitutions, deletions, and/or insertions at certain positionswithin the amino acid sequence, as compared to a native or referencesequence. Ordinarily, variants possess at least 50% identity to awild-type, native or reference sequence. In some embodiments, variantsshare at least 80%, or at least 90% identity with a wild-type, native orreference sequence.

Variant antigens/polypeptides encoded by nucleic acids of the disclosuremay contain amino acid changes that confer any of a number of desirableproperties, e.g., that enhance their immunogenicity, enhance theirexpression, and/or improve their stability or PK/PD properties in asubject. Variant antigens/polypeptides can be made using routinemutagenesis techniques and assayed as appropriate to determine whetherthey possess the desired property. Assays to determine expression levelsand immunogenicity are well known in the art and exemplary such assaysare set forth in the Examples section. Similarly, PK/PD properties of aprotein variant can be measured using art recognized techniques, e.g.,by determining expression of antigens in a vaccinated subject over timeand/or by looking at the durability of the induced immune response. Thestability of protein(s) encoded by a variant nucleic acid may bemeasured by assaying thermal stability or stability upon ureadenaturation or may be measured using in silico prediction. Methods forsuch experiments and in silico determinations are known in the art.

In some embodiments, a hMPV/hPIV3 vaccine comprises an mRNA ORF having anucleotide sequence identified by any one of the sequences providedherein (see e.g., Sequence Listing), or having a nucleotide sequence atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identical to a nucleotidesequence identified by any one of the sequence provided herein.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptides (e.g. antigens) or polynucleotides (nucleicacids), as determined by comparing the sequences. Identity also refersto the degree of sequence relatedness between or among sequences asdetermined by the number of matches between strings of two or more aminoacid residues or nucleic acid residues. Identity measures the percent ofidentical matches between the smaller of two or more sequences with gapalignments (if any) addressed by a particular mathematical model orcomputer program (e.g., “algorithms”). Identity of related antigens ornucleic acids can be readily calculated by known methods. “Percent (%)identity” as it applies to polypeptide or polynucleotide sequences isdefined as the percentage of residues (amino acid residues or nucleicacid residues) in the candidate amino acid or nucleic acid sequence thatare identical with the residues in the amino acid sequence or nucleicacid sequence of a second sequence after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent identity.Methods and computer programs for the alignment are well known in theart. It is understood that identity depends on a calculation of percentidentity but may differ in value due to gaps and penalties introduced inthe calculation. Generally, variants of a particular polynucleotide orpolypeptide (e.g., antigen) have at least 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% butless than 100% sequence identity to that particular referencepolynucleotide or polypeptide as determined by sequence alignmentprograms and parameters described herein and known to those skilled inthe art. Such tools for alignment include those of the BLAST suite(Stephen F. Altschul, et al (1997), “Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs”, Nucleic Acids Res.25:3389-3402). Another popular local alignment technique is based on theSmith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981)“Identification of common molecular subsequences.” J. Mol. Biol.147:195-197). A general global alignment technique based on dynamicprogramming is the Needleman-Wunsch algorithm (Needleman, S. B. &Wunsch, C. D. (1970) “A general method applicable to the search forsimilarities in the amino acid sequences of two proteins.” J. Mol. Biol.48:443-453). More recently a Fast Optimal Global Sequence AlignmentAlgorithm (FOGSAA) has been developed that purportedly produces globalalignment of nucleotide and protein sequences faster than other optimalglobal alignment methods, including the Needleman-Wunsch algorithm.

As such, polynucleotides encoding peptides or polypeptides containingsubstitutions, insertions and/or additions, deletions and covalentmodifications with respect to reference sequences, in particular thepolypeptide (e.g., antigen) sequences disclosed herein, are includedwithin the scope of this disclosure. For example, sequence tags or aminoacids, such as one or more lysines, can be added to peptide sequences(e.g., at the N-terminal or C-terminal ends). Sequence tags can be usedfor peptide detection, purification or localization. Lysines can be usedto increase peptide solubility or to allow for biotinylation.Alternatively, amino acid residues located at the carboxy and aminoterminal regions of the amino acid sequence of a peptide or protein mayoptionally be deleted providing for truncated sequences. Certain aminoacids (e.g., C-terminal or N-terminal residues) may alternatively bedeleted depending on the use of the sequence, as for example, expressionof the sequence as part of a larger sequence which is soluble, or linkedto a solid support. In some embodiments, sequences for (or encoding)signal sequences, termination sequences, transmembrane domains, linkers,multimerization domains (such as, e.g., foldon regions) and the like maybe substituted with alternative sequences that achieve the same or asimilar function. In some embodiments, cavities in the core of proteinscan be filled to improve stability, e.g., by introducing larger aminoacids. In other embodiments, buried hydrogen bond networks may bereplaced with hydrophobic resides to improve stability. In yet otherembodiments, glycosylation sites may be removed and replaced withappropriate residues. Such sequences are readily identifiable to one ofskill in the art. It should also be understood that some of thesequences provided herein contain sequence tags or terminal peptidesequences (e.g., at the N-terminal or C-terminal ends) that may bedeleted, for example, prior to use in the preparation of an RNA (e.g.,mRNA) vaccine.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of hMPV/hPIV3 antigens of interest. For example,provided herein is any protein fragment (meaning a polypeptide sequenceat least one amino acid residue shorter than a reference antigensequence but otherwise identical) of a reference protein, provided thatthe fragment is immunogenic and confers a protective immune response tohMPV/hPIV3. In addition to variants that are identical to the referenceprotein but are truncated, in some embodiments, an antigen includes 2,3, 4, 5, 6, 7, 8, 9, 10, or more mutations, as shown in any of thesequences provided or referenced herein. Antigens/antigenic polypeptidescan range in length from about 4, 6, or 8 amino acids to full lengthproteins.

Stabilizing Elements

Naturally-occurring eukaryotic mRNA molecules have been found to containstabilizing elements, including, but not limited to untranslated regions(UTR) at their 5′-end (5′UTR) and/or at their 3′-end (3′UTR), inaddition to other structural features, such as a 5′-cap structure or a3′-poly(A) tail. Both the 5′UTR and the 3′UTR are typically transcribedfrom the genomic DNA and are elements of the premature mRNA.Characteristic structural features of mature mRNA, such as the 5′-capand the 3′-poly(A) tail are usually added to the transcribed (premature)mRNA during mRNA processing. The 3′-poly(A) tail is typically a stretchof adenine nucleotides added to the 3′-end of the transcribed mRNA. Itcan comprise up to about 400 adenine nucleotides. In some embodimentsthe length of the 3′-poly(A) tail may be an essential element withrespect to the stability of the individual mRNA.

In some embodiments the RNA (e.g., mRNA) vaccine may include one or morestabilizing elements. Stabilizing elements may include for instance ahistone stem-loop. A stem-loop binding protein (SLBP), a 32 kDa proteinhas been identified. It is associated with the histone stem-loop at the3′-end of the histone messages in both the nucleus and the cytoplasm.Its expression level is regulated by the cell cycle; it peaks during theS-phase, when histone mRNA levels are also elevated. The protein hasbeen shown to be essential for efficient 3′-end processing of histonepre-mRNA by the U7 snRNP. SLBP continues to be associated with thestem-loop after processing, and then stimulates the translation ofmature histone mRNAs into histone proteins in the cytoplasm. The RNAbinding domain of SLBP is conserved through metazoa and protozoa; itsbinding to the histone stem-loop depends on the structure of the loop.The minimum binding site includes at least three nucleotides 5′ and twonucleotides 3′ relative to the stem-loop.

In some embodiments, a hMPV/hPIV3 RNA (e.g., mRNA) vaccine of thepresent disclosure comprises a natural 5′ cap. In some embodiments, a 5′cap may be a 5′ cap analog, such as a 5′ diguanosine cap, tetraphosphatecap analogs having a methylene-bis (phosphonate) moiety, cap analogshaving a sulfur substitution for a non-bridging oxygen, N7-benzylateddinucleoside tetraphosphate analogs, or anti-reverse cap analogs. Insome embodiments, the 5′ cap is a 7mG(5′)ppp(5′)NlmpNp cap. In someembodiments, the 5′ cap is a 7mG(5′)ppp(5′)NlmpN2mp cap. In someembodiments, the 5′cap analog is a 5′ diguanosine cap.

In some embodiments, a hMPV/hPIV3 RNA (e.g., mRNA) vaccine includes acoding region, at least one histone stem-loop, and optionally, a poly(A)sequence or polyadenylation signal. The poly(A) sequence orpolyadenylation signal generally should enhance the expression level ofthe encoded protein. The encoded protein, in some embodiments, is not ahistone protein, a reporter protein (e.g. luciferase, green fluorescentprotein (GFP), enhanced GFP (EGFP), or (3-Galactosidase), or a marker orselection protein (e.g. alpha-globin, galactokinase and xanthine:guanine phosphoribosyl transferase (GPT)).

In some embodiments, the combination of a poly(A) sequence orpolyadenylation signal and at least one histone stem-loop, even thoughboth represent alternative mechanisms in nature, acts synergistically toincrease the protein expression beyond the level observed with either ofthe individual elements. It has been found that the synergistic effectof the combination of poly(A) and at least one histone stem-loop doesnot depend on the order of the elements or the length of the poly(A)sequence.

In some embodiments, a hMPV/hPIV3 RNA (e.g., mRNA) vaccine does notcomprise a histone downstream element (HDE). “Histone downstreamelement” (HDE) includes a purine-rich polynucleotide stretch ofapproximately 15 to 20 nucleotides 3′ of naturally occurring stem-loops,representing the binding site for the U7 snRNA, which is involved inprocessing of histone pre-mRNA into mature histone mRNA. Ideally, theinventive nucleic acid does not include an intron.

In some embodiments, a hMPV/hPIV3 RNA (e.g., mRNA) vaccine may or maynot contain a enhancer and/or promoter sequence, which may be modifiedor unmodified or which may be activated or inactivated. In someembodiments, the histone stem-loop is generally derived from histonegenes, and includes an intramolecular base pairing of two neighboredpartially or entirely reverse complementary sequences separated by aspacer, including (e.g., consisting of) a short sequence, which formsthe loop of the structure. The unpaired loop region is typically unableto base pair with either of the stem loop elements. It occurs more oftenin RNA, as is a key component of many RNA secondary structures, but maybe present in single-stranded DNA as well. Stability of the stem-loopstructure generally depends on the length, number of mismatches orbulges, and base composition of the paired region. In some embodiments,wobble base pairing (non-Watson-Crick base pairing) may result. In someembodiments, the at least one histone stem-loop sequence comprises alength of 15 to 45 nucleotides.

In other embodiments a hMPV/hPIV3 RNA (e.g., mRNA) vaccine may have oneor more AU-rich sequences removed. These sequences, sometimes referredto as AURES are destabilizing sequences found in the 3′UTR. The AURESmay be removed from the RNA (e.g., mRNA) vaccines. Alternatively theAURES may remain in the RNA (e.g., mRNA) vaccine.

Signal Peptides

In some embodiments, a hMPV/hPIV3 vaccine comprises a RNA having an ORFthat encodes a signal peptide fused to the hMPV/hPIV3 antigen. Signalpeptides, comprising the N-terminal 15-60 amino acids of proteins, aretypically needed for the translocation across the membrane on thesecretory pathway and, thus, universally control the entry of mostproteins both in eukaryotes and prokaryotes to the secretory pathway. Ineukaryotes, the signal peptide of a nascent precursor protein(pre-protein) directs the ribosome to the rough endoplasmic reticulum(ER) membrane and initiates the transport of the growing peptide chainacross it for processing. ER processing produces mature proteins,wherein the signal peptide is cleaved from precursor proteins, typicallyby a ER-resident signal peptidase of the host cell, or they remainuncleaved and function as a membrane anchor. A signal peptide may alsofacilitate the targeting of the protein to the cell membrane.

A signal peptide may have a length of 15-60 amino acids. For example, asignal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,or 60 amino acids. In some embodiments, a signal peptide has a length of20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55, 20-55,25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50,35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40,20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30,25-30, 15-25, 20-25, or 15-20 amino acids.

Signal peptides from heterologous genes (which regulate expression ofgenes other tha hMPV/hPIV3 antigens in nature) are known in the art andcan be tested for desired properties and then incorporated into anucleic acid of the disclosure. In some embodiments, the signal peptideis a bovine prolactin signal peptide. For example, the bovine prolactinsignal peptide may comprise sequence MDSKGSSQKGSRLLLLLVVSNLLLPQGVVG (SEQID NO: 17). Other signal peptide sequences may also be used. Forexample, the signal peptide may comprise one of the following sequences:MDWTWILFLVAAATRVHS (SEQ ID NO: 18); METPAQLLFLLLLWLPDTTG (SEQ ID NO:19); MLGSNSGQRVVFTILLLLVAPAYS (SEQ ID NO: 20); MKCLLYLAFLFIGVNCA (SEQ IDNO: 21); MWLVSLAIVTACAGA (SEQ ID NO: 22).

Fusion Proteins

In some embodiments, a hMPV/hPIV3 RNA vaccine of the present disclosureincludes an RNA encoding an antigenic fusion protein. Thus, the encodedantigen or antigens may include two or more proteins (e.g., proteinand/or protein fragment) joined together. Alternatively, the protein towhich a protein antigen is fused does not promote a strong immuneresponse to itself, but rather to the hMPV/hPIV3 antigen. Antigenicfusion proteins, in some embodiments, retain the functional propertyfrom each original protein.

Scaffold Moieties

The RNA (e.g., mRNA) vaccines as provided herein, in some embodiments,encode fusion proteins which comprise hMPV/hPIV3 antigens linked toscaffold moieties. In some embodiments, such scaffold moieties impartdesired properties to an antigen encoded by a nucleic acid of thedisclosure. For example scaffold proteins may improve the immunogenicityof an antigen, e.g., by altering the structure of the antigen, alteringthe uptake and processing of the antigen, and/or causing the antigen tobind to a binding partner.

In some embodiments, the scaffold moiety is protein that canself-assemble into protein nanoparticles that are highly symmetric,stable, and structurally organized, with diameters of 10-150 nm, ahighly suitable size range for optimal interactions with various cellsof the immune system. In some embodiments, viral proteins or virus-likeparticles can be used to form stable nanoparticle structures. Examplesof such viral proteins are known in the art. For example, in someembodiments, the scaffold moiety is a hepatitis B surface antigen(HBsAg). HBsAg forms spherical particles with an average diameter of ˜22nm and which lacked nucleic acid and hence are non-infectious(Lopez-Sagaseta, J. et al. Computational and Structural BiotechnologyJournal 14 (2016) 58-68). In some embodiments, the scaffold moiety is ahepatitis B core antigen (HBcAg) self-assembles into particles of 24-31nm diameter, which resembled the viral cores obtained from HBV-infectedhuman liver. HBcAg produced in self-assembles into two classes ofdifferently sized nanoparticles of 300 Å and 360 Å diameter,corresponding to 180 or 240 protomers. In some embodiments a hMPV/hPIV3antigen is fused to HBsAG or HBcAG to facilitate self-assembly ofnanoparticles displaying the hMPV/hPIV3 antigen.

In another embodiment, bacterial protein platforms may be used.Non-limiting examples of these self-assembling proteins includeferritin, lumazine and encapsulin.

Ferritin is a protein whose main function is intracellular iron storage.Ferritin is made of 24 subunits, each composed of a four-alpha-helixbundle, that self-assemble in a quaternary structure with octahedralsymmetry (Cho K. J. et al. J Mol Biol. 2009; 390:83-98). Severalhigh-resolution structures of ferritin have been determined, confirmingthat Helicobacter pylori ferritin is made of 24 identical protomers,whereas in animals, there are ferritin light and heavy chains that canassemble alone or combine with different ratios into particles of 24subunits (Granier T. et al. J Boil Inorg Chem. 2003; 8:105-111; LawsonD. M. et al. Nature. 1991; 349:541-544). Ferritin self-assembles intonanoparticles with robust thermal and chemical stability. Thus, theferritin nanoparticle is well-suited to carry and expose antigens.

Lumazine synthase (LS) is also well-suited as a nanoparticle platformfor antigen display. LS, which is responsible for the penultimatecatalytic step in the biosynthesis of riboflavin, is an enzyme presentin a broad variety of organisms, including archaea, bacteria, fungi,plants, and eubacteria (Weber S. E. Flavins and Flavoproteins. Methodsand Protocols, Series: Methods in Molecular Biology. 2014). The LSmonomer is 150 amino acids long, and consists of beta-sheets along withtandem alpha-helices flanking its sides. A number of differentquaternary structures have been reported for LS, illustrating itsmorphological versatility: from homopentamers up to symmetricalassemblies of 12 pentamers forming capsids of 150 Å diameter. Even LScages of more than 100 subunits have been described (Zhang X. et al. JMol Biol. 2006; 362:753-770).

Encapsulin, a novel protein cage nanoparticle isolated from thermophileThermotoga maritima, may also be used as a platform to present antigenson the surface of self-assembling nanoparticles. Encapsulin is assembledfrom 60 copies of identical 31 kDa monomers having a thin andicosahedral T=1 symmetric cage structure with interior and exteriordiameters of 20 and 24 nm, respectively (Sutter M. et al. Nat Struct MolBiol. 2008, 15: 939-947). Although the exact function of encapsulin inT. maritima is not clearly understood yet, its crystal structure hasbeen recently solved and its function was postulated as a cellularcompartment that encapsulates proteins such as DyP (Dye decolorizingperoxidase) and Flp (Ferritin like protein), which are involved inoxidative stress responses (Rahmanpour R. et al. FEBS J. 2013, 280:2097-2104).

Linkers and Cleavable Peptides

In some embodiments, the mRNAs of the disclosure encode more than onepolypeptide, referred to herein as fusion proteins. In some embodiments,the mRNA further encodes a linker located between at least one or eachdomain of the fusion protein. The linker can be, for example, acleavable linker or protease-sensitive linker. In some embodiments, thelinker is selected from the group consisting of F2A linker, P2A linker,T2A linker, E2A linker, and combinations thereof. This family ofself-cleaving peptide linkers, referred to as 2A peptides, has beendescribed in the art (see for example, Kim, J. H. et al. (2011) PLoS ONE6:e18556). In some embodiments, the linker is an F2A linker. In someembodiments, the linker is a GGGS linker. In some embodiments, thefusion protein contains three domains with intervening linkers, havingthe structure: domain-linker-domain-linker-domain.

Cleavable linkers known in the art may be used in connection with thedisclosure. Exemplary such linkers include: F2A linkers,T2A linkers, P2Alinkers, E2A linkers (See, e.g., WO2017127750). The skilled artisan willappreciate that other art-recognized linkers may be suitable for use inthe constructs of the disclosure (e.g., encoded by the nucleic acids ofthe disclosure). The skilled artisan will likewise appreciate that otherpolycistronic constructs (mRNA encoding more than oneantigen/polypeptide separately within the same molecule) may be suitablefor use as provided herein.

Sequence Optimization

In some embodiments, an ORF encoding an antigen of the disclosure iscodon optimized. Codon optimization methods are known in the art. Forexample, an ORF of any one or more of the sequences provided herein maybe codon optimized. Codon optimization, in some embodiments, may be usedto match codon frequencies in target and host organisms to ensure properfolding; bias GC content to increase mRNA stability or reduce secondarystructures; minimize tandem repeat codons or base runs that may impairgene construction or expression; customize transcriptional andtranslational control regions; insert or remove protein traffickingsequences; remove/add post translation modification sites in encodedprotein (e.g., glycosylation sites); add, remove or shuffle proteindomains; insert or delete restriction sites; modify ribosome bindingsites and mRNA degradation sites; adjust translational rates to allowthe various domains of the protein to fold properly; or reduce oreliminate problem secondary structures within the polynucleotide. Codonoptimization tools, algorithms and services are known in theart—non-limiting examples include services from GeneArt (LifeTechnologies), DNA2.0 (Menlo Park Calif.) and/or proprietary methods. Insome embodiments, the open reading frame (ORF) sequence is optimizedusing optimization algorithms.

In some embodiments, a codon optimized sequence shares less than 95%sequence identity to a naturally-occurring or wild-type sequence ORF(e.g., a naturally-occurring or wild-type mRNA sequence encoding ahMPV/hPIV3 antigen). In some embodiments, a codon optimized sequenceshares less than 90% sequence identity to a naturally-occurring orwild-type sequence (e.g., a naturally-occurring or wild-type mRNAsequence encoding a hMPV/hPIV3 antigen). In some embodiments, a codonoptimized sequence shares less than 85% sequence identity to anaturally-occurring or wild-type sequence (e.g., a naturally-occurringor wild-type mRNA sequence encoding a hMPV/hPIV3 antigen). In someembodiments, a codon optimized sequence shares less than 80% sequenceidentity to a naturally-occurring or wild-type sequence (e.g., anaturally-occurring or wild-type mRNA sequence encoding a hMPV/hPIV3antigen). In some embodiments, a codon optimized sequence shares lessthan 75% sequence identity to a naturally-occurring or wild-typesequence (e.g., a naturally-occurring or wild-type mRNA sequenceencoding a hMPV/hPIV3 antigen).

In some embodiments, a codon optimized sequence shares between 65% and85% (e.g., between about 67% and about 85% or between about 67% andabout 80%) sequence identity to a naturally-occurring or wild-typesequence (e.g., a naturally-occurring or wild-type mRNA sequenceencoding a hMPV/hPIV3 antigen). In some embodiments, a codon optimizedsequence shares between 65% and 75% or about 80% sequence identity to anaturally-occurring or wild-type sequence (e.g., a naturally-occurringor wild-type mRNA sequence encoding a hMPV/hPIV3 antigen).

In some embodiments, a codon-optimized sequence encodes an antigen thatis as immunogenic as, or more immunogenic than (e.g., at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 100%, orat least 200% more), than a hMPV/hPIV3 antigen encoded by anon-codon-optimized sequence.

When transfected into mammalian host cells, the modified mRNAs have astability of between 12-18 hours, or greater than 18 hours, e.g., 24,36, 48, 60, 72, or greater than 72 hours and are capable of beingexpressed by the mammalian host cells.

In some embodiments, a codon optimized RNA may be one in which thelevels of G/C are enhanced. The G/C-content of nucleic acid molecules(e.g., mRNA) may influence the stability of the RNA. RNA having anincreased amount of guanine (G) and/or cytosine (C) residues may befunctionally more stable than RNA containing a large amount of adenine(A) and thymine (T) or uracil (U) nucleotides. As an example,WO02/098443 discloses a pharmaceutical composition containing an mRNAstabilized by sequence modifications in the translated region. Due tothe degeneracy of the genetic code, the modifications work bysubstituting existing codons for those that promote greater RNAstability without changing the resulting amino acid. The approach islimited to coding regions of the RNA.

Chemically Unmodified Nucleotides

In some embodiments, at least one RNA (e.g., mRNA) of a hMPV/hPIV3vaccines of the present disclosure is not chemically modified andcomprises the standard ribonucleotides consisting of adenosine,guanosine, cytosine and uridine. In some embodiments, nucleotides andnucleosides of the present disclosure comprise standard nucleosideresidues such as those present in transcribed RNA (e.g. A, G, C, or U).In some embodiments, nucleotides and nucleosides of the presentdisclosure comprise standard deoxyribonucleosides such as those presentin DNA (e.g. dA, dG, dC, or dT).

Chemical Modifications

hMPV/hPIV3 RNA vaccines of the present disclosure comprise, in someembodiments, at least one nucleic acid (e.g., RNA) having an openreading frame encoding at least one hMPV/hPIV3 antigen, wherein thenucleic acid comprises nucleotides and/or nucleosides that can bestandard (unmodified) or modified as is known in the art. In someembodiments, nucleotides and nucleosides of the present disclosurecomprise modified nucleotides or nucleosides. Such modified nucleotidesand nucleosides can be naturally-occurring modified nucleotides andnucleosides or non-naturally occurring modified nucleotides andnucleosides. Such modifications can include those at the sugar,backbone, or nucleobase portion of the nucleotide and/or nucleoside asare recognized in the art.

In some embodiments, a naturally-occurring modified nucleotide ornucleotide of the disclosure is one as is generally known or recognizedin the art. Non-limiting examples of such naturally occurring modifiednucleotides and nucleotides can be found, inter alia, in the widelyrecognized MODOMICS database.

In some embodiments, a non-naturally occurring modified nucleotide ornucleoside of the disclosure is one as is generally known or recognizedin the art. Non-limiting examples of such non-naturally occurringmodified nucleotides and nucleosides can be found, inter alia, inpublished US application Nos. PCT/US2012/058519; PCT/US2013/075177;PCT/US2014/058897; PCT/U52014/058891; PCT/U52014/070413;PCT/US2015/36773; PCT/US2015/36759; PCT/US2015/36771; orPCT/I132017/051367 all of which are incorporated by reference herein.

Hence, nucleic acids of the disclosure (e.g., DNA nucleic acids and RNAnucleic acids, such as mRNA nucleic acids) can comprise standardnucleotides and nucleosides, naturally-occurring nucleotides andnucleosides, non-naturally-occurring nucleotides and nucleosides, or anycombination thereof.

Nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleicacids, such as mRNA nucleic acids), in some embodiments, comprisevarious (more than one) different types of standard and/or modifiednucleotides and nucleosides. In some embodiments, a particular region ofa nucleic acid contains one, two or more (optionally different) types ofstandard and/or modified nucleotides and nucleosides.

In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNAnucleic acid), introduced to a cell or organism, exhibits reduceddegradation in the cell or organism, respectively, relative to anunmodified nucleic acid comprising standard nucleotides and nucleosides.

In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNAnucleic acid), introduced into a cell or organism, may exhibit reducedimmunogenicity in the cell or organism, respectively (e.g., a reducedinnate response) relative to an unmodified nucleic acid comprisingstandard nucleotides and nucleosides.

Nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids), insome embodiments, comprise non-natural modified nucleotides that areintroduced during synthesis or post-synthesis of the nucleic acids toachieve desired functions or properties. The modifications may bepresent on internucleotide linkages, purine or pyrimidine bases, orsugars. The modification may be introduced with chemical synthesis orwith a polymerase enzyme at the terminal of a chain or anywhere else inthe chain. Any of the regions of a nucleic acid may be chemicallymodified.

The present disclosure provides for modified nucleosides and nucleotidesof a nucleic acid (e.g., RNA nucleic acids, such as mRNA nucleic acids).A “nucleoside” refers to a compound containing a sugar molecule (e.g., apentose or ribose) or a derivative thereof in combination with anorganic base (e.g., a purine or pyrimidine) or a derivative thereof(also referred to herein as “nucleobase”). A “nucleotide” refers to anucleoside, including a phosphate group. Modified nucleotides may bysynthesized by any useful method, such as, for example, chemically,enzymatically, or recombinantly, to include one or more modified ornon-natural nucleosides. Nucleic acids can comprise a region or regionsof linked nucleosides. Such regions may have variable backbone linkages.The linkages can be standard phosphodiester linkages, in which case thenucleic acids would comprise regions of nucleotides.

Modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures, such as, forexample, in those nucleic acids having at least one chemicalmodification. One example of such non-standard base pairing is the basepairing between the modified nucleotide inosine and adenine, cytosine oruracil. Any combination of base/sugar or linker may be incorporated intonucleic acids of the present disclosure.

In some embodiments, modified nucleobases in nucleic acids (e.g., RNAnucleic acids, such as mRNA nucleic acids) comprise1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ),5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine(ψ). In some embodiments, modified nucleobases in nucleic acids (e.g.,RNA nucleic acids, such as mRNA nucleic acids) comprise 5-methoxymethyluridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methylcytidine, and/or 5-methoxy cytidine. In some embodiments, thepolyribonucleotide includes a combination of at least two (e.g., 2, 3, 4or more) of any of the aforementioned modified nucleobases, includingbut not limited to chemical modifications.

In some embodiments, a RNA nucleic acid of the disclosure comprises1-methyl-pseudouridine (m1ψ) substitutions at one or more or all uridinepositions of the nucleic acid.

In some embodiments, a RNA nucleic acid of the disclosure comprises1-methyl-pseudouridine (m1ψ) substitutions at one or more or all uridinepositions of the nucleic acid and 5-methyl cytidine substitutions at oneor more or all cytidine positions of the nucleic acid.

In some embodiments, a RNA nucleic acid of the disclosure comprisespseudouridine (ψ) substitutions at one or more or all uridine positionsof the nucleic acid.

In some embodiments, a RNA nucleic acid of the disclosure comprisespseudouridine (ψ) substitutions at one or more or all uridine positionsof the nucleic acid and 5-methyl cytidine substitutions at one or moreor all cytidine positions of the nucleic acid.

In some embodiments, a RNA nucleic acid of the disclosure comprisesuridine at one or more or all uridine positions of the nucleic acid.

In some embodiments, nucleic acids (e.g., RNA nucleic acids, such asmRNA nucleic acids) are uniformly modified (e.g., fully modified,modified throughout the entire sequence) for a particular modification.For example, a nucleic acid can be uniformly modified with1-methyl-pseudouridine, meaning that all uridine residues in the mRNAsequence are replaced with 1-methyl-pseudouridine. Similarly, a nucleicacid can be uniformly modified for any type of nucleoside residuepresent in the sequence by replacement with a modified residue such asthose set forth above.

The nucleic acids of the present disclosure may be partially or fullymodified along the entire length of the molecule. For example, one ormore or all or a given type of nucleotide (e.g., purine or pyrimidine,or any one or more or all of A, G, U, C) may be uniformly modified in anucleic acid of the disclosure, or in a predetermined sequence regionthereof (e.g., in the mRNA including or excluding the polyA tail). Insome embodiments, all nucleotides X in a nucleic acid of the presentdisclosure (or in a sequence region thereof) are modified nucleotides,wherein X may be any one of nucleotides A, G, U, C, or any one of thecombinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

The nucleic acid may contain from about 1% to about 100% modifiednucleotides (either in relation to overall nucleotide content, or inrelation to one or more types of nucleotide, i.e., any one or more of A,G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1%to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%,from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10%to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%,from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%,from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%,from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%,from 90% to 100%, and from 95% to 100%). It will be understood that anyremaining percentage is accounted for by the presence of unmodified A,G, U, or C.

The nucleic acids may contain at a minimum 1% and at maximum 100%modified nucleotides, or any intervening percentage, such as at least 5%modified nucleotides, at least 10% modified nucleotides, at least 25%modified nucleotides, at least 50% modified nucleotides, at least 80%modified nucleotides, or at least 90% modified nucleotides. For example,the nucleic acids may contain a modified pyrimidine such as a modifieduracil or cytosine. In some embodiments, at least 5%, at least 10%, atleast 25%, at least 50%, at least 80%, at least 90% or 100% of theuracil in the nucleic acid is replaced with a modified uracil (e.g., a5-substituted uracil). The modified uracil can be replaced by a compoundhaving a single unique structure, or can be replaced by a plurality ofcompounds having different structures (e.g., 2, 3, 4 or more uniquestructures). In some embodiments, at least 5%, at least 10%, at least25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine inthe nucleic acid is replaced with a modified cytosine (e.g., a5-substituted cytosine). The modified cytosine can be replaced by acompound having a single unique structure, or can be replaced by aplurality of compounds having different structures (e.g., 2, 3, 4 ormore unique structures).

N-Linked Glycosylation Site Mutants

N-linked glycans of viral proteins play important roles in modulatingthe immune response. Glycans can be important for maintaining theappropriate antigenic conformations, shielding potential neutralizationepitopes, and may alter the proteolytic susceptibility of proteins. Someviruses have putative N-linked glycosylation sites. Deletion ormodification of an N-linked glycosylation site may enhance the immuneresponse. Thus, the present disclosure provides, in some embodiments,hMPV/hPIV3 RNA (e.g., mRNA) vaccines comprising nucleic acids (e.g.,mRNA) encoding antigenic polypeptides (e.g., hMPV/hPIV3 F proteins) thatcomprise a deletion or modification at one or more N-linkedglycosylation sites.

Untranslated Regions (UTRs)

The nucleic acids of the present disclosure may comprise one or moreregions or parts which act or function as an untranslated region. Wherenucleic acids are designed to encode at least one antigen of interest,the nucleic may comprise one or more of these untranslated regions(UTRs). Wild-type untranslated regions of a nucleic acid are transcribedbut not translated. In mRNA, the 5′ UTR starts at the transcriptionstart site and continues to the start codon but does not include thestart codon; whereas, the 3′ UTR starts immediately following the stopcodon and continues until the transcriptional termination signal. Thereis growing body of evidence about the regulatory roles played by theUTRs in terms of stability of the nucleic acid molecule and translation.The regulatory features of a UTR can be incorporated into thepolynucleotides of the present disclosure to, among other things,enhance the stability of the molecule. The specific features can also beincorporated to ensure controlled down-regulation of the transcript incase they are misdirected to undesired organs sites. A variety of 5′UTRand 3′UTR sequences are known and available in the art.

A 5′ UTR is region of an mRNA that is directly upstream (5′) from thestart codon (the first codon of an mRNA transcript translated by aribosome). A 5′ UTR does not encode a protein (is non-coding). Natural5′UTRs have features that play roles in translation initiation. Theyharbor signatures like Kozak sequences which are commonly known to beinvolved in the process by which the ribosome initiates translation ofmany genes. Kozak sequences have the consensus CCR(A/G)CCAUGG (SEQ IDNO: 23), where R is a purine (adenine or guanine) three bases upstreamof the start codon (AUG), which is followed by another ‘G’.5′UTR alsohave been known to form secondary structures which are involved inelongation factor binding.

In some embodiments of the disclosure, a 5′ UTR is a heterologous UTR,i.e., is a UTR found in nature associated with a different ORF. Inanother embodiment, a 5′ UTR is a synthetic UTR, i.e., does not occur innature. Synthetic UTRs include UTRs that have been mutated to improvetheir properties, e.g., which increase gene expression as well as thosewhich are completely synthetic. Exemplary 5′ UTRs include Xenopus orhuman derived a-globin or b-globin (8278063; 9012219), human cytochromeb-245 a polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobaccoetch virus (U.S. Pat. Nos. 8,278,063, 9,012,219). CMV immediate-early 1(IE1) gene (US20140206753, WO2013/185069), the sequence GGGAUCCUACC (SEQID NO: 24) (WO2014/144196) may also be used. In another embodiment, 5′UTR of a TOP gene is a 5′ UTR of a TOP gene lacking the 5′ TOP motif(the oligopyrimidine tract) (e.g., WO2015/101414, WO2015/101415,WO2015/062738, WO2015/024667, WO2015/024667; 5′ UTR element derived fromribosomal protein Large 32 (L32) gene (WO2015/101414, WO2015/101415,WO2015/062738), 5′ UTR element derived from the 5′UTR of anhydroxysteroid (17-β) dehydrogenase 4 gene (HSD17B4) (WO2015/024667), ora 5′ UTR element derived from the 5′ UTR of ATP5A1 (OW2015/024667) canbe used. In some embodiments, an internal ribosome entry site (IRES) isused instead of a 5′ UTR.

In some embodiments, a 5′ UTR of the present disclosure comprises thesequence of SEQ ID NO: 12.

A 3′ UTR is region of an mRNA that is directly downstream (3′) from thestop codon (the codon of an mRNA transcript that signals a terminationof translation). A 3′ UTR does not encode a protein (is non-coding).Natural or wild type 3′ UTRs are known to have stretches of adenosinesand uridines embedded in them. These AU rich signatures are particularlyprevalent in genes with high rates of turnover. Based on their sequencefeatures and functional properties, the AU rich elements (AREs) can beseparated into three classes (Chen et al, 1995): Class I AREs containseveral dispersed copies of an AUUUA motif within U-rich regions. C-Mycand MyoD contain class I AREs. Class II AREs possess two or moreoverlapping UUAUUUA(U/A)(U/A) (SEQ ID NO: 25) nonamers. Moleculescontaining this type of AREs include GM-CSF and TNF-a. Class III ARESare less well defined. These U rich regions do not contain an AUUUAmotif c-Jun and Myogenin are two well-studied examples of this class.Most proteins binding to the AREs are known to destabilize themessenger, whereas members of the ELAV family, most notably HuR, havebeen documented to increase the stability of mRNA. HuR binds to AREs ofall the three classes. Engineering the HuR specific binding sites intothe 3′ UTR of nucleic acid molecules will lead to HuR binding and thus,stabilization of the message in vivo.

Introduction, removal or modification of 3′ UTR AU rich elements (AREs)can be used to modulate the stability of nucleic acids (e.g., RNA) ofthe disclosure. When engineering specific nucleic acids, one or morecopies of an ARE can be introduced to make nucleic acids of thedisclosure less stable and thereby curtail translation and decreaseproduction of the resultant protein. Likewise, AREs can be identifiedand removed or mutated to increase the intracellular stability and thusincrease translation and production of the resultant protein.Transfection experiments can be conducted in relevant cell lines, usingnucleic acids of the disclosure and protein production can be assayed atvarious time points post-transfection. For example, cells can betransfected with different ARE-engineering molecules and by using anELISA kit to the relevant protein and assaying protein produced at 6hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection.

3′ UTRs may be heterologous or synthetic. With respect to 3′ UTRs,globin UTRs, including Xenopus β-globin UTRs and human β-globin UTRs areknown in the art (8278063, 9012219, US20110086907). A modified β-globinconstruct with enhanced stability in some cell types by cloning twosequential human β-globin 3′UTRs head to tail has been developed and iswell known in the art (US2012/0195936, WO2014/071963). In additiona2-globin, a1-globin, UTRs and mutants thereof are also known in the art(WO2015/101415, WO2015/024667). Other 3′ UTRs described in the mRNAconstructs in the non-patent literature include CYBA (Ferizi et al.,2015) and albumin (Thess et al., 2015). Other exemplary 3′ UTRs includethat of bovine or human growth hormone (wild type or modified)(WO2013/185069, US20140206753, WO2014/152774), rabbit β globin andhepatitis B virus (HBV), α-globin 3′ UTR and Viral VEEV 3′ UTR sequencesare also known in the art. In some embodiments, the sequence UUUGAAUU(WO2014/144196) is used. In some embodiments, 3′ UTRs of human and mouseribosomal protein are used. Other examples include rps9 3′UTR(WO2015/101414), FIG4 (WO2015/101415), and human albumin 7(WO2015/101415).

In some embodiments, a 3′ UTR of the present disclosure comprises thesequence of SEQ ID NO: 13.

Those of ordinary skill in the art will understand that 5′UTRs that areheterologous or synthetic may be used with any desired 3′ UTR sequence.For example, a heterologous 5′UTR may be used with a synthetic 3′UTRwith a heterologous 3″ UTR.

Non-UTR sequences may also be used as regions or subregions within anucleic acid. For example, introns or portions of introns sequences maybe incorporated into regions of nucleic acid of the disclosure.Incorporation of intronic sequences may increase protein production aswell as nucleic acid levels.

Combinations of features may be included in flanking regions and may becontained within other features. For example, the ORF may be flanked bya 5′ UTR which may contain a strong Kozak translational initiationsignal and/or a 3′ UTR which may include an oligo(dT) sequence fortemplated addition of a poly-A tail. 5′ UTR may comprise a firstpolynucleotide fragment and a second polynucleotide fragment from thesame and/or different genes such as the 5′ UTRs described in US PatentApplication Publication No.20100293625 and PCT/US2014/069155, hereinincorporated by reference in its entirety.

It should be understood that any UTR from any gene may be incorporatedinto the regions of a nucleic acid. Furthermore, multiple wild-type UTRsof any known gene may be utilized. It is also within the scope of thepresent disclosure to provide artificial UTRs which are not variants ofwild type regions. These UTRs or portions thereof may be placed in thesame orientation as in the transcript from which they were selected ormay be altered in orientation or location. Hence a 5′ or 3′ UTR may beinverted, shortened, lengthened, made with one or more other 5′ UTRs or3′ UTRs. As used herein, the term “altered” as it relates to a UTRsequence, means that the UTR has been changed in some way in relation toa reference sequence. For example, a 3′ UTR or 5′ UTR may be alteredrelative to a wild-type or native UTR by the change in orientation orlocation as taught above or may be altered by the inclusion ofadditional nucleotides, deletion of nucleotides, swapping ortransposition of nucleotides. Any of these changes producing an“altered” UTR (whether 3′ or 5′) comprise a variant UTR.

In some embodiments, a double, triple or quadruple UTR such as a 5′ UTRor 3′ UTR may be used. As used herein, a “double” UTR is one in whichtwo copies of the same UTR are encoded either in series or substantiallyin series. For example, a double beta-globin 3′ UTR may be used asdescribed in US Patent publication 2010/0129877, the contents of whichare incorporated herein by reference in its entirety.

It is also within the scope of the present disclosure to have patternedUTRs. As used herein “patterned UTRs” are those UTRs which reflect arepeating or alternating pattern, such as ABABAB or AABBAABBAABB orABCABCABC or variants thereof repeated once, twice, or more than 3times. In these patterns, each letter, A, B, or C represent a differentUTR at the nucleotide level.

In some embodiments, flanking regions are selected from a family oftranscripts whose proteins share a common function, structure, featureor property. For example, polypeptides of interest may belong to afamily of proteins which are expressed in a particular cell, tissue orat some time during development. The UTRs from any of these genes may beswapped for any other UTR of the same or different family of proteins tocreate a new polynucleotide. As used herein, a “family of proteins” isused in the broadest sense to refer to a group of two or morepolypeptides of interest which share at least one function, structure,feature, localization, origin, or expression pattern.

The untranslated region may also include translation enhancer elements(TEE). As a non-limiting example, the TEE may include those described inUS patent publication 2009/0226470, herein incorporated by reference inits entirety, and those known in the art.

In Vitro Transcription of RNA (e.g., mRNA)

A hMPV/hPIV3 RNA (e.g., mRNA) vaccine of the present disclosure compriseat least one RNA polynucleotide, such as a mRNA (e.g., modified mRNA).mRNA, for example, is transcribed in vitro from template DNA, referredto as an “in vitro transcription template.” In some embodiments, an invitro transcription template encodes a 5′ untranslated (UTR) region,contains an open reading frame, and encodes a 3′ UTR and a polyA tail.The particular nucleic acid sequence composition and length of an invitro transcription template will depend on the mRNA encoded by thetemplate.

In some embodiments, the in vitro transcription template used to producethe RNA (e.g., mRNA) polynucleotides of the present disclosure comprisesthe nucleotide sequence identified by any one of SEQ ID NO:1-3.

A “5′ untranslated region” (5′UTR) refers to a region of an mRNA that isdirectly upstream (i.e., 5′) from the start codon (i.e., the first codonof an mRNA transcript translated by a ribosome) that does not encode apolypeptide.

A “3′ untranslated region” (3′UTR) refers to a region of an mRNA that isdirectly downstream (i.e., 3′) from the stop codon (i.e., the codon ofan mRNA transcript that signals a termination of translation) that doesnot encode a polypeptide.

An “open reading frame” is a continuous stretch of DNA beginning with astart codon (e.g., methionine (ATG)), and ending with a stop codon(e.g., TAA, TAG or TGA) and encodes a polypeptide.

A “polyA tail” is a region of mRNA that is downstream, e.g., directlydownstream (i.e., 3′), from the 3′ UTR that contains multiple,consecutive adenosine monophosphates. A polyA tail may contain 10 to 300adenosine monophosphates. For example, a polyA tail may contain 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosinemonophosphates. In some embodiments, a polyA tail contains 50 to 250adenosine monophosphates. In a relevant biological setting (e.g., incells, in vivo) the poly(A) tail functions to protect mRNA fromenzymatic degradation, e.g., in the cytoplasm, and aids in transcriptiontermination, export of the mRNA from the nucleus and translation.

In some embodiments, a polynucleotide includes 200 to 3,000 nucleotides.For example, a polynucleotide may include 200 to 500, 200 to 1000, 200to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to3000 nucleotides.

Chemical Synthesis

Solid-phase chemical synthesis. Nucleic acids the present disclosure maybe manufactured in whole or in part using solid phase techniques.Solid-phase chemical synthesis of nucleic acids is an automated methodwherein molecules are immobilized on a solid support and synthesizedstep by step in a reactant solution. Solid-phase synthesis is useful insite-specific introduction of chemical modifications in the nucleic acidsequences.

Liquid Phase Chemical Synthesis. The synthesis of nucleic acids of thepresent disclosure by the sequential addition of monomer building blocksmay be carried out in a liquid phase.

Combination of Synthetic Methods. The synthetic methods discussed aboveeach has its own advantages and limitations. Attempts have beenconducted to combine these methods to overcome the limitations. Suchcombinations of methods are within the scope of the present disclosure.The use of solid-phase or liquid-phase chemical synthesis in combinationwith enzymatic ligation provides an efficient way to generate long chainnucleic acids that cannot be obtained by chemical synthesis alone.

Ligation of Nucleic Acid Regions or Subregions

Assembling nucleic acids by a ligase may also be used. DNA or RNAligases promote intermolecular ligation of the 5′ and 3′ ends ofpolynucleotide chains through the formation of a phosphodiester bond.Nucleic acids such as chimeric polynucleotides and/or circular nucleicacids may be prepared by ligation of one or more regions or subregions.DNA fragments can be joined by a ligase catalyzed reaction to createrecombinant DNA with different functions. Two oligodeoxynucleotides, onewith a 5′ phosphoryl group and another with a free 3′ hydroxyl group,serve as substrates for a DNA ligase.

Purification

Purification of the nucleic acids described herein may include, but isnot limited to, nucleic acid clean-up, quality assurance and qualitycontrol. Clean-up may be performed by methods known in the arts such as,but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers,Mass.), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc,Vedbaek, Denmark) or HPLC based purification methods such as, but notlimited to, strong anion exchange HPLC, weak anion exchange HPLC,reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC(HIC-HPLC). The term “purified” when used in relation to a nucleic acidsuch as a “purified nucleic acid” refers to one that is separated fromat least one contaminant. A “contaminant” is any substance that makesanother unfit, impure or inferior. Thus, a purified nucleic acid (e.g.,DNA and RNA) is present in a form or setting different from that inwhich it is found in nature, or a form or setting different from thatwhich existed prior to subjecting it to a treatment or purificationmethod.

A quality assurance and/or quality control check may be conducted usingmethods such as, but not limited to, gel electrophoresis, UV absorbance,or analytical HPLC.

In some embodiments, the nucleic acids may be sequenced by methodsincluding, but not limited to reverse-transcriptase-PCR.

Quantification

In some embodiments, the nucleic acids of the present disclosure may bequantified in exosomes or when derived from one or more bodily fluid.Bodily fluids include peripheral blood, serum, plasma, ascites, urine,cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid,aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolarlavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatoryfluid, sweat, fecal matter, hair, tears, cyst fluid, pleural andperitoneal fluid, pericardial fluid, lymph, chyme, chyle, bile,interstitial fluid, menses, pus, sebum, vomit, vaginal secretions,mucosal secretion, stool water, pancreatic juice, lavage fluids fromsinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, andumbilical cord blood. Alternatively, exosomes may be retrieved from anorgan selected from the group consisting of lung, heart, pancreas,stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast,prostate, brain, esophagus, liver, and placenta.

Assays may be performed using construct specific probes, cytometry,qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, massspectrometry, or combinations thereof while the exosomes may be isolatedusing immunohistochemical methods such as enzyme linked immunosorbentassay (ELISA) methods. Exosomes may also be isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.

These methods afford the investigator the ability to monitor, in realtime, the level of nucleic acids remaining or delivered. This ispossible because the nucleic acids of the present disclosure, in someembodiments, differ from the endogenous forms due to the structural orchemical modifications.

In some embodiments, the nucleic acid may be quantified using methodssuch as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).A non-limiting example of a UV/Vis spectrometer is a NANODROP®spectrometer (ThermoFisher, Waltham, Mass.). The quantified nucleic acidmay be analyzed in order to determine if the nucleic acid may be ofproper size, check that no degradation of the nucleic acid has occurred.Degradation of the nucleic acid may be checked by methods such as, butnot limited to, agarose gel electrophoresis, HPLC based purificationmethods such as, but not limited to, strong anion exchange HPLC, weakanion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobicinteraction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry(LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis(CGE).

Pharmaceutical Formulations

Provided herein are compositions (e.g., pharmaceutical compositions),methods, kits and reagents for prevention or treatment of hMPV/hPIV3 inhumans and other mammals, for example. hMPV/hPIV3 RNA (e.g., mRNA)vaccines can be used as therapeutic or prophylactic agents. They may beused in medicine to prevent and/or treat infectious disease.

In some embodiments, a hMPV/hPIV3 vaccine containing RNA polynucleotidesas described herein can be administered to a subject (e.g., a mammaliansubject, such as a human subject), and the RNA polynucleotides aretranslated in vivo to produce an antigenic polypeptide (antigen).

An “effective amount” of a hMPV/hPIV3 vaccine is based, at least inpart, on the target tissue, target cell type, means of administration,physical characteristics of the RNA (e.g., length, nucleotidecomposition, and/or extent of modified nucleosides), other components ofthe vaccine, and other determinants, such as age, body weight, height,sex and general health of the subject. Typically, an effective amount ofa hMPV/hPIV3 vaccine provides an induced or boosted immune response as afunction of antigen production in the cells of the subject. In someembodiments, an effective amount of the hMPV/hPIV3 RNA vaccinecontaining RNA polynucleotides having at least one chemicalmodifications are more efficient than a composition containing acorresponding unmodified polynucleotide encoding the same antigen or apeptide antigen. Increased antigen production may be demonstrated byincreased cell transfection (the percentage of cells transfected withthe RNA vaccine), increased protein translation and/or expression fromthe polynucleotide, decreased nucleic acid degradation (as demonstrated,for example, by increased duration of protein translation from amodified polynucleotide), or altered antigen specific immune response ofthe host cell.

In some embodiments, a vaccine of the present disclosure is demonstratedto be effective by showing a desired result in an animal model, e.g., arodent or non-human primate model. For example, vaccination of cottonrats with a hMPV/hPIV3 combination vaccine as provided herein resultedin the induction of high levels of neutralizing antibodies and reducedthe viral titers in the nose and lungs of the immunized cotton ratsafter challenge with hMPV or hPIV3 viruses, without evidence ofvaccine-enhanced respiratory disease (ERD). Studies of vaccinatedAfrican Green Monkeys demonstrated similar results. The hMPV/PIV3combination vaccine afforded full protection against both viruses in thelung and the nose of the vaccinated animals after less than threeintramuscular doses of the vaccine.

The term “pharmaceutical composition” refers to the combination of anactive agent with a carrier, inert or active, making the compositionespecially suitable for diagnostic or therapeutic use in vivo or exvivo. A “pharmaceutically acceptable carrier,” after administered to orupon a subject, does not cause undesirable physiological effects. Thecarrier in the pharmaceutical composition must be “acceptable” also inthe sense that it is compatible with the active ingredient and can becapable of stabilizing it. One or more solubilizing agents can beutilized as pharmaceutical carriers for delivery of an active agent.Examples of a pharmaceutically acceptable carrier include, but are notlimited to, biocompatible vehicles, adjuvants, additives, and diluentsto achieve a composition usable as a dosage form. Examples of othercarriers include colloidal silicon oxide, magnesium stearate, cellulose,and sodium lauryl sulfate. Additional suitable pharmaceutical carriersand diluents, as well as pharmaceutical necessities for their use, aredescribed in Remington's Pharmaceutical Sciences.

In some embodiments, RNA vaccines (including polynucleotides and theirencoded polypeptides) in accordance with the present disclosure may beused for treatment or prevention of hMPV/hPIV3. hMPV/hPIV3 RNA vaccinesmay be administered prophylactically or therapeutically as part of anactive immunization scheme to healthy individuals or early in infectionduring the incubation phase or during active infection after onset ofsymptoms. In some embodiments, the amount of RNA vaccines of the presentdisclosure provided to a cell, a tissue or a subject may be an amounteffective for immune prophylaxis.

hMPV/hPIV3 RNA (e.g., mRNA) vaccines may be administered with otherprophylactic or therapeutic compounds. As a non-limiting example, aprophylactic or therapeutic compound may be an adjuvant or a booster. Asused herein, when referring to a prophylactic composition, such as avaccine, the term “booster” refers to an extra administration of theprophylactic (vaccine) composition. A booster (or booster vaccine) maybe given after an earlier administration of the prophylacticcomposition. The time of administration between the initialadministration of the prophylactic composition and the booster may be,but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years,7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95years or more than 99 years. In exemplary embodiments, the time ofadministration between the initial administration of the prophylacticcomposition and the booster may be, but is not limited to, 1 week, 2weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months or 1 year.

In some embodiments, hMPV/hPIV3 RNA vaccines may be administeredintramuscularly, intranasally or intradermally, similarly to theadministration of inactivated vaccines known in the art.

The hMPV/hPIV3 RNA vaccines may be utilized in various settingsdepending on the prevalence of the infection or the degree or level ofunmet medical need. As a non-limiting example, the RNA vaccines may beutilized to treat and/or prevent a variety of infectious disease. RNAvaccines have superior properties in that they produce much largerantibody titers, better neutralizing immunity, produce more durableimmune responses, and/or produce responses earlier than commerciallyavailable vaccines.

Provided herein are pharmaceutical compositions including hMPV/hPIV3 RNAvaccines and RNA vaccine compositions and/or complexes optionally incombination with one or more pharmaceutically acceptable excipients.

hMPV/hPIV3 RNA (e.g., mRNA) vaccines may be formulated or administeredalone or in conjunction with one or more other components. For instance,hMPV/hPIV3 RNA vaccines (vaccine compositions) may comprise othercomponents including, but not limited to, adjuvants.

In some embodiments, hMPV/hPIV3 RNA vaccines do not include an adjuvant(they are adjuvant free).

hMPV/hPIV3 RNA (e.g., mRNA) vaccines may be formulated or administeredin combination with one or more pharmaceutically-acceptable excipients.In some embodiments, vaccine compositions comprise at least oneadditional active substances, such as, for example, atherapeutically-active substance, a prophylactically-active substance,or a combination of both. Vaccine compositions may be sterile,pyrogen-free or both sterile and pyrogen-free. General considerations inthe formulation and/or manufacture of pharmaceutical agents, such asvaccine compositions, may be found, for example, in Remington: TheScience and Practice of Pharmacy 21st ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference in its entirety).

In some embodiments, hMPV/hPIV3 RNA vaccines are administered to humans,human patients or subjects. For the purposes of the present disclosure,the phrase “active ingredient” generally refers to the RNA vaccines orthe polynucleotides contained therein, for example, RNA polynucleotides(e.g., mRNA polynucleotides) encoding antigens.

Formulations of the vaccine compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient (e.g., mRNA polynucleotide) intoassociation with an excipient and/or one or more other accessoryingredients, and then, if necessary and/or desirable, dividing, shapingand/or packaging the product into a desired single- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

In some embodiments, hMPV/hPIV3 RNA vaccines are formulated using one ormore excipients to: (1) increase stability; (2) increase celltransfection; (3) permit the sustained or delayed release (e.g., from adepot formulation); (4) alter the biodistribution (e.g., target tospecific tissues or cell types); (5) increase the translation of encodedprotein in vivo; and/or (6) alter the release profile of encoded protein(antigen) in vivo. In addition to traditional excipients such as any andall solvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, excipients can include,without limitation, lipidoids, liposomes, lipid nanoparticles, polymers,lipoplexes, core-shell nanoparticles, peptides, proteins, cellstransfected with hMPV/hPIV3 RNA vaccines (e.g., for transplantation intoa subject), hyaluronidase, nanoparticle mimics and combinations thereof.

Flagellin Adjuvants

Flagellin is an approximately 500 amino acid monomeric protein thatpolymerizes to form the flagella associated with bacterial motion.Flagellin is expressed by a variety of flagellated bacteria (Salmonellatyphimurium for example) as well as non-flagellated bacteria (such asEscherichia coli). Sensing of flagellin by cells of the innate immunesystem (dendritic cells, macrophages, etc.) is mediated by the Toll-likereceptor 5 (TLR5) as well as by Nod-like receptors (NLRs) Ipaf andNaip5. TLRs and NLRs have been identified as playing a role in theactivation of innate immune response and adaptive immune response. Assuch, flagellin provides an adjuvant effect in a vaccine.

The nucleotide and amino acid sequences encoding known flagellinpolypeptides are publicly available in the NCBI GenBank database. Theflagellin sequences from S. Typhimurium, H. Pylori, V. Cholera, S.marcesens, S. flexneri, T. Pallidum, L. pneumophila, B. burgdorferei, C.difficile, R. meliloti, A. tumefaciens, R. lupini, B. clarridgeiae, P.Mirabilis, B. subtilus, L. monocytogenes, P. aeruginosa, and E. coli,among others are known.

A flagellin polypeptide, as used herein, refers to a full lengthflagellin protein, immunogenic fragments thereof, and peptides having atleast 50% sequence identify to a flagellin protein or immunogenicfragments thereof. Exemplary flagellin proteins include flagellin fromSalmonella typhi (UniPro Entry number: Q56086), Salmonella typhimurium(A0A0C9DG09), Salmonella enteritidis (A0A0C9BAB7), and Salmonellacholeraesuis (Q6V2X8). In some embodiments, the flagellin proteincomprises the following amino acid sequence:MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANGTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKEISSKTLGLDKLNVQDAYTPKETAVTVDKTTYKNGTDPITAQSNTDIQTAIGGGATGVTGADIKFKDGQYYLDVKGGASAGVYKATYDETTKKVNIDTTDKTPLATAEATAIRGTATITHNQIAEVTKEGVDTTTVAAQLAAAGVTGADKDNTSLVKLSFEDKNGKVIDGGYAVKMGDDFYAATYDEKTGAITAKTTTYTDGTGVAQTGAVKFGGANGKSEVVTATDGKTYLASDLDKHNFRTGGELKEVNTDKTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLSSARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLS LLR (SEQ IDNO:10). In some embodiments, the flagellin polypeptide has at least 60%,70%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identify to aflagellin protein or immunogenic fragments thereof.

In some embodiments, the flagellin polypeptide is an immunogenicfragment. An immunogenic fragment is a portion of a flagellin proteinthat provokes an immune response. In some embodiments, the immuneresponse is a TLRS immune response. An example of an immunogenicfragment is a flagellin protein in which all or a portion of a hingeregion has been deleted or replaced with other amino acids. For example,an antigenic polypeptide may be inserted in the hinge region. Hingeregions are the hypervariable regions of a flagellin. Hinge regions of aflagellin are also referred to as “D3 domain or region, “propellerdomain or region,” “hypervariable domain or region” and “variable domainor region.” “At least a portion of a hinge region,” as used herein,refers to any part of the hinge region of the flagellin, or the entiretyof the hinge region. In other embodiments an immunogenic fragment offlagellin is a 20, 25, 30, 35, or 40 amino acid C-terminal fragment offlagellin.

The flagellin monomer is formed by domains D0 through D3. D0 and D1,which form the stem, are composed of tandem long alpha helices and arehighly conserved among different bacteria. The D1 domain includesseveral stretches of amino acids that are useful for TLR5 activation.The entire D1 domain or one or more of the active regions within thedomain are immunogenic fragments of flagellin. Examples of immunogenicregions within the D1 domain include residues 88-114 and residues411-431 (in Salmonella typhimurium FliC flagellin. Within the 13 aminoacids in the 88-100 region, at least 6 substitutions are permittedbetween Salmonella flagellin and other flagellins that still preserveTLR5 activation. Thus, immunogenic fragments of flagellin includeflagellin like sequences that activate TLRS and contain a 13 amino acidmotif that is 53% or more identical to the Salmonella sequence in 88-100of FliC (LQRVRELAVQSAN; SEQ ID NO:11).

In some embodiments, the RNA (e.g., mRNA) vaccine includes an RNA thatencodes a fusion protein of flagellin and one or more antigenicpolypeptides. A “fusion protein” as used herein, refers to a linking oftwo components of the construct. In some embodiments, a carboxy-terminusof the antigenic polypeptide is fused or linked to an amino terminus ofthe flagellin polypeptide. In other embodiments, an amino-terminus ofthe antigenic polypeptide is fused or linked to a carboxy-terminus ofthe flagellin polypeptide. The fusion protein may include, for example,one, two, three, four, five, six or more flagellin polypeptides linkedto one, two, three, four, five, six or more antigenic polypeptides. Whentwo or more flagellin polypeptides and/or two or more antigenicpolypeptides are linked such a construct may be referred to as a“multimer.”

Each of the components of a fusion protein may be directly linked to oneanother or they may be connected through a linker. For instance, thelinker may be an amino acid linker. The amino acid linker encoded for bythe RNA (e.g., mRNA) vaccine to link the components of the fusionprotein may include, for instance, at least one member selected from thegroup consisting of a lysine residue, a glutamic acid residue, a serineresidue and an arginine residue. In some embodiments the linker is 1-30,1-25, 1-25, 5-10, 5, 15, or 5-20 amino acids in length.

In other embodiments the RNA (e.g., mRNA) vaccine includes at least twoseparate RNA polynucleotides, one encoding one or more antigenicpolypeptides (e.g., F proteins) and the other encoding the flagellinpolypeptide. The at least two RNA polynucleotides may be co-formulatedin a carrier such as a lipid nanoparticle. Alternatively, the at leasttwo RNA polynucleotides may be separately formulated.

Lipid Nanoparticles (LNPs) In some embodiments, hMPV/hPIV3 RNA (e.g.,mRNA) vaccines of the disclosure are formulated in a lipid nanoparticle(LNP). Lipid nanoparticles typically comprise ionizable cationic lipid,non-cationic lipid, sterol and PEG lipid components along with thenucleic acid cargo of interest. The lipid nanoparticles of thedisclosure can be generated using components, compositions, and methodsas are generally known in the art, see for example PCT/US2016/052352;PCT/US2016/068300; PCT/US2017/037551; PCT/US2015/027400;PCT/US2016/047406; PCT/US2016/000129; PCT/US2016/014280;PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077;PCT/US2014/055394; PCT/US2016/52117; PCT/US2012/069610;PCT/US2017/027492; PCT/US2016/059575 and PCT/US2016/069491 all of whichare incorporated by reference herein in their entirety.

Vaccines of the present disclosure are typically formulated in lipidnanoparticle. In some embodiments, the lipid nanoparticle comprises atleast one ionizable cationic lipid, at least one non-cationic lipid, atleast one sterol, and/or at least one polyethylene glycol (PEG)-modifiedlipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of20-60% ionizable cationic lipid. For example, the lipid nanoparticle maycomprise a molar ratio of 20-50%, 20-40%, 20-30%, 30-60%, 30-50%,30-40%, 40-60%, 40-50%, or 50-60% ionizable cationic lipid. In someembodiments, the lipid nanoparticle comprises a molar ratio of 20%, 30%,40%, 50, or 60% ionizable cationic lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of5-25% non-cationic lipid. For example, the lipid nanoparticle maycomprise a molar ratio of 5-20%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%,15-25%, 15-20%, or 20-25% non-cationic lipid. In some embodiments, thelipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, or25%non-cationic lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of25-55% sterol. For example, the lipid nanoparticle may comprise a molarratio of 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30-50%, 30-45%,30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%,45-55%, 45-50%, or 50-55% sterol. In some embodiments, the lipidnanoparticle comprises a molar ratio of 25%, 30%, 35%, 40%, 45%, 50%, or55% sterol.

In some embodiments, the lipid nanoparticle comprises a molar ratio of0.5-15% PEG-modified lipid. For example, the lipid nanoparticle maycomprise a molar ratio of 0.5-10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%,2-10%, 2-5%, 5-15%, 5-10%, or 10-15%. In some embodiments, the lipidnanoparticle comprises a molar ratio of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% PEG-modified lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55%sterol, and 0.5-15% PEG-modified lipid.

In some embodiments, an ionizable cationic lipid of the disclosurecomprises a compound of Formula (I):

or a salt or isomer thereof, wherein:

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH2)_(n)Q, —(CH2)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a carbocycle, heterocycle, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —N(R)₂,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,—N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

In some embodiments, a subset of compounds of Formula (I) includes thosein which when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then(i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or7-membered heterocycloalkyl when n is 1 or 2.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O) N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and a 5- to14-membered heterocycloalkyl having one or more heteroatoms selectedfrom N, O, and S which is substituted with one or more substituentsselected from oxo (═O), OH, amino, mono- or di-alkylamino, and C₁₋₃alkyl, and each n is independently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheterocycle having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(═NR₉)N(R)₂, and eachn is independently selected from 1, 2, 3, 4, and 5; and when Q is a 5-to 14-membered heterocycle and (i) R₄ is —(CH₂)_(n)Q in which n is 1 or2, or (ii) R₄ is —(CH₂)_(n)CHQR in which n is 1, or (iii) R₄ is —CHQR,and —CQ(R)₂, then Q is either a 5- to 14-membered heteroaryl or 8- to14-membered heterocycloalkyl;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, andunsubstituted C₁₋₆ alkyl, where Q is selected from a C₃₋₆ carbocycle, a5- to 14-membered heteroaryl having one or more heteroatoms selectedfrom N, O, and S, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H,—CXH₂, —CN, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂,—N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR,—N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR,—N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,—N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R,—C(O)N(R)OR, and —C(═NR₉)N(R)₂, and each n is independently selectedfrom 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is —N(R)₂, and n isselected from 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′; R₂ and R₃ are independently selected from thegroup consisting of C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and—R*OR″, or R₂ and R₃, together with the atom to which they are attached,form a heterocycle or carbocycle;

R₄ is selected from the group consisting of —(CH₂)_(n)Q, —(CH₂)_(n)CHQR,—CHQR, and —CQ(R)₂, where Q is —N(R)₂, and n is selected from 1, 2, 3,4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IA):

or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M′; R₄ isunsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroarylor heterocycloalkyl; M and M′ are independently selected from —C(O)O—,—OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and aheteroaryl group; and R₂ and R₃ are independently selected from thegroup consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (II):

or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and5; M₁ is a bond or M′; R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q,in which n is 2, 3, or 4, and Q is OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈, —NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M′ areindependently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—,—S—S—, an aryl group, and a heteroaryl group; and R₂ and R₃ areindependently selected from the group consisting of H, C₁₋₁₄ alkyl, andC₂₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IIa), (IIb), (IIc), or (IIe):

or a salt or isomer thereof, wherein R₄ is as described herein.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IId):

or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R′, R″, andR₂ through R₆ are as described herein. For example, each of R₂ and R₃may be independently selected from the group consisting of C₅₋₁₄ alkyland C₅₋₁₄ alkenyl.

In some embodiments, an ionizable cationic lipid of the disclosurecomprises a compound having structure:

In some embodiments, an ionizable cationic lipid of the disclosurecomprises a compound having structure:

In some embodiments, a non-cationic lipid of the disclosure comprises1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine,1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG),sphingomyelin, and mixtures thereof.

In some embodiments, a PEG modified lipid of the disclosure comprises aPEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid,a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modifieddiacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof Insome embodiments, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (alsoreferred to as PEG-DOMG), PEG-DSG and/or PEG-DPG.

In some embodiments, a sterol of the disclosure comprises cholesterol,fecosterol, sitosterol, ergosterol, campesterol, stigmasterol,brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixturesthereof.

In some embodiments, a LNP of the disclosure comprises an ionizablecationic lipid of Compound 1, wherein the non-cationic lipid is DSPC,the structural lipid that is cholesterol, and the PEG lipid is PEG-DMG.

In some embodiments, a LNP of the disclosure comprises a mixture of 4lipids, including Compound 1; 1,2-dimyristoyl-sn-glycerol,methoxypolyethyleneglycol (PEG2000-DMG);1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and cholesterol. Insome embodiments, a vaccine comprises a mRNA encoding a hMPV F proteinof strain A/TN92-4 (e.g., SEQ ID NO:4) and a mRNA encoding a PIV3 Fprotein of strain PER/FLA4815/2008 (e.g., SEQ ID NO:5) formulated in aLNP that comprises a mixture of 4 lipids, including Compound 1;1,2-dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG2000-DMG);DSPC; and cholesterol.

In some embodiments, a LNP of the disclosure comprises an N:P ratio offrom about 2:1 to about 30:1.

In some embodiments, a LNP of the disclosure comprises an N:P ratio ofabout 6:1.

In some embodiments, a LNP of the disclosure comprises an N:P ratio ofabout 3:1.

In some embodiments, a LNP of the disclosure comprises a wt/wt ratio ofthe ionizable cationic lipid component to the RNA of from about 10:1 toabout 100:1.

In some embodiments, a LNP of the disclosure comprises a wt/wt ratio ofthe ionizable cationic lipid component to the RNA of about 20:1.

In some embodiments, a LNP of the disclosure comprises a wt/wt ratio ofthe ionizable cationic lipid component to the RNA of about 10:1.

In some embodiments, a LNP of the disclosure has a mean diameter fromabout 50 nm to about 150 nm.

In some embodiments, a LNP of the disclosure has a mean diameter fromabout 70 nm to about 120 nm.

Multivalent Vaccines

The hMPV/hPIV3 vaccines, as provided herein, may include an RNA (e.g.mRNA) or multiple RNAs encoding two or more antigens of the same ordifferent hMPV/hPIV3 species. In some embodiments, a hMPV/hPIV3 vaccineincludes an RNA or multiple RNAs encoding two or more antigens selectedfrom hMPV F protein and hPIVs F protein. In some embodiments, the RNA(at least one RNA) of a hMPV/hPIV3 vaccine may encode 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, or more antigens.

In some embodiments, two or more different RNA (e.g., mRNA) encodingantigens may be formulated in the same lipid nanoparticle. In otherembodiments, two or more different RNA encoding antigens may beformulated in separate lipid nanoparticles (each RNA formulated in asingle lipid nanoparticle). The lipid nanoparticles may then be combinedand administered as a single vaccine composition (e.g., comprisingmultiple RNA encoding multiple antigens) or may be administeredseparately.

Combination Vaccines

The hMPV/hPIV3 vaccines, as provided herein, may include an RNA ormultiple RNAs encoding two or more antigens of the same or differenthMPV/hPIV3 strains. Also provided herein are combination vaccines thatinclude RNA encoding one or more hMPV/hPIV3 antigen(s) and one or moreantigen(s) of a different organisms (e.g., bacterial and/or viralorganism). For example, in some embodiments, an hMPV/PIV3 vaccine of thepresent disclosure comprises an hMPV fusion (F) protein and a PIV3 Fprotein. In some embodiments, the two F proteins are co-formulated at a1:1 mass ratio in an LNP. In some embodiments, the two F proteins areformulated separately in separate LNPs. Thus, the vaccines of thepresent disclosure may be combination vaccines that target one or moreantigens of the same strain/species, or one or more antigens ofdifferent strains/species, e.g., antigens which induce immunity toorganisms which are found in the same geographic areas where the risk ofhMPV/hPIV3 infection is high or organisms to which an individual islikely to be exposed to when exposed to hMPV/hPIV3. In some embodiments,the hMPV F protein is of the A/TN92-4 hMPV strain and the hPIV3 Fprotein is of the PER/FLA4815/2008 strain.

Dosing/Administration

Provided herein are compositions (e.g., pharmaceutical compositions),methods, kits and reagents for prevention and/or treatment of hMPV/hPIV3in humans and other mammals. hMPV/hPIV3 RNA vaccines can be used astherapeutic or prophylactic agents. In some aspects, the RNA vaccines ofthe disclosure are used to provide prophylactic protection fromhMPV/hPIV3. In some aspects, the RNA vaccines of the disclosure are usedto treat a hMPV/hPIV3 infection. In some embodiments, the hMPV/hPIV3vaccines of the present disclosure are used in the priming of immuneeffector cells, for example, to activate peripheral blood mononuclearcells (PBMCs) ex vivo, which are then infused (re-infused) into asubject.

A subject may be any mammal, including non-human primate and humansubjects. Typically, a subject is a human subject.

In some embodiments, the hMPV/hPIV3 vaccines are administered to asubject (e.g., a mammalian subject, such as a human subject) in aneffective amount to induce an antigen-specific immune response. The RNAencoding the hMPV/hPIV3 antigen is expressed and translated in vivo toproduce the antigen, which then stimulates an immune response in thesubject.

In some embodiments, an hMPV/hPIV3 vaccine of the disclosure results inhigh neutralizing antibody titers (e.g., as measured by the 60%reduction end point assay) after fewer than three doses (e.g., after oneor two doses). In some embodiments, a cotton rat model can be used totest the vaccine and a titer of at least 9 (log 2 transformed titerusing 60% PRNT assay) can be measured using this assay. In someembodiments, a vaccine of the disclosure results in a higher titer thanthat observed with a traditional formalin inactivated protein vaccine(e.g., FI-PIV3 or FI-HMPV). In another embodiment, an hMPV/hPIV3 vaccineof the disclosure protects against challenge infection, e.g., asmeasured by reduced viral load in both the nose and lung after fewerthan three doses. In some embodiments, a cotton rat model can be used totest this end point. Surprisingly, an hMPV/hPIV3 vaccine of thedisclosure results in neutralizing antibody titers and reduced viralload, but does not result in alveolitis or interstitial pneumonia. Insome embodiments, a cotton rat model can be used to determine lungpathology. In another embodiment, the protection provided by anhMPV/hPIV3 vaccine of the disclosure protects against challenge withHPIV3 even though PIV3-HN mRNA is not present in the vaccine.

Prophylactic protection from hMPV/hPIV3 can be achieved followingadministration of a hMPV/hPIV3 RNA vaccine of the present disclosure.Vaccines can be administered once, twice, three times, four times ormore but it is likely sufficient to administer the vaccine once(optionally followed by a single booster). It is possible, although lessdesirable, to administer the vaccine to an infected individual toachieve a therapeutic response. Dosing may need to be adjustedaccordingly.

A method of eliciting an immune response in a subject against hMPV/hPIV3is provided in aspects of the present disclosure. The method involvesadministering to the subject a hMPV/hPIV3 RNA vaccine comprising atleast one RNA (e.g., mRNA) having an open reading frame encoding atleast one hMPV/hPIV3 antigen, thereby inducing in the subject an immuneresponse specific to hMPV/hPIV3 antigen, wherein anti-antigen antibodytiter in the subject is increased following vaccination relative toanti-antigen antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against thehMPV/hPIV3. An “anti-antigen antibody” is a serum antibody the bindsspecifically to the antigen.

A prophylactically effective dose is an effective dose that preventsinfection with the virus at a clinically acceptable level. In someembodiments, the effective dose is a dose listed in a package insert forthe vaccine. A traditional vaccine, as used herein, refers to a vaccineother than the mRNA vaccines of the present disclosure. For instance, atraditional vaccine includes, but is not limited, to live microorganismvaccines, killed microorganism vaccines, subunit vaccines, proteinantigen vaccines, DNA vaccines, virus like particle (VLP) vaccines, etc.In exemplary embodiments, a traditional vaccine is a vaccine that hasachieved regulatory approval and/or is registered by a national drugregulatory body, for example the Food and Drug Administration (FDA) inthe United States or the European Medicines Agency (EMA).

In some embodiments, the anti-antigen antibody titer in the subject isincreased 1 log to 10 log following vaccination relative to anti-antigenantibody titer in a subject vaccinated with a prophylactically effectivedose of a traditional vaccine against the hMPV/hPIV3 or an unvaccinatedsubject. In some embodiments, the anti-antigen antibody titer in thesubject is increased 1 log, 2 log, 3 log, 4 log, 5 log, or 10 logfollowing vaccination relative to anti-antigen antibody titer in asubject vaccinated with a prophylactically effective dose of atraditional vaccine against the hMPV/hPIV3 or an unvaccinated subject.

A method of eliciting an immune response in a subject against ahMPV/hPIV3 is provided in other aspects of the disclosure. The methodinvolves administering to the subject a hMPV/hPIV3 RNA vaccinecomprising at least one RNA polynucleotide having an open reading frameencoding at least one hMPV/hPIV3 antigen, thereby inducing in thesubject an immune response specific to hMPV/hPIV3 antigen, wherein theimmune response in the subject is higher than or equivalent to an immuneresponse in a subject vaccinated with a traditional vaccine against thehMPV/hPIV3. In some embodiments, the response is higher for the mRNAvaccine of the disclosure even when the protein vaccine is administeredat 2 times to 100 times the dosage level relative to the RNA vaccine.

In some embodiments, the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine attwice the dosage level relative to the hMPV/hPIV3 RNA vaccine. In someembodiments, the immune response in the subject is equivalent to animmune response in a subject vaccinated with a traditional vaccine atthree times the dosage level relative to the hMPV/hPIV3 RNA vaccine. Insome embodiments, the immune response in the subject is equivalent to animmune response in a subject vaccinated with a traditional vaccine at 4times, 5 times, 10 times, 50 times, or 100 times the dosage levelrelative to the hMPV/hPIV3 RNA vaccine. In some embodiments, the immuneresponse in the subject is equivalent to an immune response in a subjectvaccinated with a traditional vaccine at 10 times to 1000 times thedosage level relative to the hMPV/hPIV3 RNA vaccine. In someembodiments, the immune response in the subject is equivalent to animmune response in a subject vaccinated with a traditional vaccine at100 times to 1000 times the dosage level relative to the hMPV/hPIV3 RNAvaccine.

In other embodiments, the immune response is assessed by determining[protein] antibody titer in the subject. In other embodiments, theability of serum or antibody from an immunized subject is tested for itsability to neutralize viral uptake or reduce hMPV/hPIV3 transformationof human B lymphocytes. In other embodiments, the ability to promote arobust T cell response(s) is measured using art recognized techniques.

Other aspects the disclosure provide methods of eliciting an immuneresponse in a subject against a hMPV/hPIV3 by administering to thesubject a hMPV/hPIV3 RNA vaccine comprising at least one RNApolynucleotide having an open reading frame encoding at least onehMPV/hPIV3 antigen, thereby inducing in the subject an immune responsespecific to hMPV/hPIV3 antigen, wherein the immune response in thesubject is induced 2 days to 10 weeks earlier relative to an immuneresponse induced in a subject vaccinated with a prophylacticallyeffective dose of a traditional vaccine against the hMPV/hPIV3. In someembodiments, the immune response in the subject is induced in a subjectvaccinated with a prophylactically effective dose of a traditionalvaccine at 2 times to 100 times the dosage level relative to the RNAvaccine.

In some embodiments, the immune response in the subject is inducedwithin 14 days of vaccine administration. In some embodiments, theimmune response in the subject increases (e.g., by at least 50%) overthe course of 14 to 27 days, 14 to 56 days, or 27 to 56 days followingvaccination.

In some embodiments, the immune response in the subject is induced 2days, 3 days, 1 week, 2 weeks, 3 weeks, 5 weeks, or 10 weeks earlierrelative to an immune response induced in a subject vaccinated with aprophylactically effective dose of a traditional vaccine.

Also provided herein are methods of eliciting an immune response in asubject against a hMPV/hPIV3 by administering to the subject ahMPV/hPIV3 RNA vaccine having an open reading frame encoding a firstantigen, wherein the RNA polynucleotide does not include a stabilizationelement, and wherein an adjuvant is not co-formulated or co-administeredwith the vaccine.

hMPV/hPIV3 RNA (e.g., mRNA) vaccines may be administered by any routewhich results in a therapeutically effective outcome. These include, butare not limited, to intradermal, intramuscular, intranasal, and/orsubcutaneous administration. The present disclosure provides methodscomprising administering RNA vaccines to a subject in need thereof. Theexact amount required will vary from subject to subject, depending onthe species, age, and general condition of the subject, the severity ofthe disease, the particular composition, its mode of administration, itsmode of activity, and the like. hMPV/hPIV3 RNA (e.g., mRNA) vaccinescompositions are typically formulated in dosage unit form for ease ofadministration and uniformity of dosage. It will be understood, however,that the total daily usage of hMPV/hPIV3 RNA (e.g., mRNA)vaccinescompositions may be decided by the attending physician within the scopeof sound medical judgment. The specific therapeutically effective,prophylactically effective, or appropriate imaging dose level for anyparticular patient will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts.

The effective amount of a hMPV/hPIV3 vaccine, as provided herein, may beas low as 20 μg, administered for example as a single dose or as two 10μg doses. In some embodiments, the effective amount is a total dose of20 μg-200 μg. For example, the effective amount may be a total dose of20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 110 μg, 120 μg, 130 μg,140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg or 200 μg. In someembodiments, the effective amount is a total dose of 25 μg-200 μg. Insome embodiments, the effective amount is a total dose of 50 μg-200 μg.

In some embodiments, hMPV/hPIV3 RNA (e.g., mRNA) vaccines compositionsmay be administered at dosage levels sufficient to deliver 0.0001 mg/kgto 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005 mg/kg to 0.05 mg/kg,0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kg to 0.5 mg/kg, 0.01 mg/kg to 50mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kg to 30 mg/kg, 0.01 mg/kg to 10mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kg to 25 mg/kg, of subject bodyweight per day, one or more times a day, per week, per month, etc. toobtain the desired therapeutic, diagnostic, prophylactic, or imagingeffect (see e.g., the range of unit doses described in InternationalPublication No. WO2013078199, herein incorporated by reference in itsentirety). The desired dosage may be delivered three times a day, twotimes a day, once a day, every other day, every third day, every week,every two weeks, every three weeks, every four weeks, every 2 months,every three months, every 6 months, etc. In certain embodiments, thedesired dosage may be delivered using multiple administrations (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, or more administrations). When multipleadministrations are employed, split dosing regimens such as thosedescribed herein may be used. In exemplary embodiments, hMPV/hPIV3 RNA(e.g., mRNA) vaccines compositions may be administered at dosage levelssufficient to deliver 0.0005 mg/kg to 0.01 mg/kg, e.g., about 0.0005mg/kg to about 0.0075 mg/kg, e.g., about 0.0005 mg/kg, about 0.001mg/kg, about 0.002 mg/kg, about 0.003 mg/kg, about 0.004 mg/kg or about0.005 mg/kg.

In some embodiments, hMPV/hPIV3 RNA (e.g., mRNA) vaccine compositionsmay be administered once or twice (or more) at dosage levels sufficientto deliver 0.025 mg/kg to 0.250 mg/kg, 0.025 mg/kg to 0.500 mg/kg, 0.025mg/kg to 0.750 mg/kg, or 0.025 mg/kg to 1.0 mg/kg.

In some embodiments, hMPV/hPIV3 RNA (e.g., mRNA) vaccine compositionsmay be administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 monthslater, Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12months later, Day 0 and 18 months later, Day 0 and 2 years later, Day 0and 5 years later, or Day 0 and 10 years later) at a total dose of or atdosage levels sufficient to deliver a total dose of 0.0100 mg, 0.025 mg,0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg,0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg,0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg,0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg,0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg,0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher and lower dosages andfrequency of administration are encompassed by the present disclosure.For example, a hMPV/hPIV3 RNA (e.g., mRNA) vaccine composition may beadministered three or four times.

In some embodiments, hMPV/hPIV3 RNA (e.g., mRNA) vaccine compositionsmay be administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 monthslater, Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12months later, Day 0 and 18 months later, Day 0 and 2 years later, Day 0and 5 years later, or Day 0 and 10 years later) at a total dose of or atdosage levels sufficient to deliver a total dose of 0.010 mg, 0.025 mg,0.100 mg or 0.400 mg.

In some embodiments, the hMPV/hPIV3 RNA (e.g., mRNA) vaccine for use ina method of vaccinating a subject is administered the subject a singledosage of between 10 μg/kg and 400 μg/kg of the nucleic acid vaccine inan effective amount to vaccinate the subject. In some embodiments, theRNA vaccine for use in a method of vaccinating a subject is administeredthe subject a single dosage of between 10 μg and 400 μg of the nucleicacid vaccine in an effective amount to vaccinate the subject. In someembodiments, a hMPV/hPIV3 RNA (e.g., mRNA) vaccine for use in a methodof vaccinating a subject is administered to the subject as a singledosage of 25-1000 μg (e.g., a single dosage of mRNA encoding ahMPV/hPIV3 antigen). In some embodiments, a hMPV/hPIV3 RNA vaccine isadministered to the subject as a single dosage of 25, 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950 or 1000 μg. For example, a hMPV/hPIV3 RNA vaccine may beadministered to a subject as a single dose of 25-100, 25-500, 50-100,50-500, 50-1000, 100-500, 100-1000, 250-500, 250-1000, or 500-1000 μg.In some embodiments, a hMPV/hPIV3 RNA (e.g., mRNA) vaccine for use in amethod of vaccinating a subject is administered to the subject as twodosages, the combination of which equals 25-1000 μg of the hMPV/hPIV3RNA (e.g., mRNA) vaccine.

A hMPV/hPIV3 RNA (e.g., mRNA) vaccine pharmaceutical compositiondescribed herein can be formulated into a dosage form described herein,such as an intranasal, intratracheal, or injectable (e.g., intravenous,intraocular, intravitreal, intramuscular, intradermal, intracardiac,intraperitoneal, and subcutaneous).

Methods of Treatment

Provided herein are compositions (e.g., pharmaceutical compositions),methods, kits and reagents for prevention and/or treatment of hMPVand/or hPIV3 infections. The RNA (e.g. mRNA) vaccines can be used astherapeutic or prophylactic agents, alone or in combination with othervaccine(s). They may be used in medicine to prevent and/or treatrespiratory disease/infection (e.g., lower respiratory hMPV/hPIV3infection). In some embodiments, the RNA (e.g., mRNA) vaccines of thepresent disclosure are used to provide prophylactic protection from hMPVand/or hPIV3. Prophylactic protection can be achieved followingadministration of a RNA (e.g., mRNA) vaccine of the present disclosure.RNA (e.g., mRNA) vaccines of the present disclosure may be used to treator prevent viral “co-infections” containing two or more respiratoryinfections. Vaccines can be administered once, twice, three times, fourtimes or more, but it is likely sufficient to administer the vaccineonce (optionally followed by a single booster). It is possible, althoughless desirable, to administer the vaccine to an infected individual toachieve a therapeutic response. Dosing may need to be adjustedaccordingly.

A method of eliciting an immune response in a subject against hMPV/hPIV3is provided in aspects of the present disclosure. The method involvesadministering to the subject an effective amount of a RNA (e.g., mRNA)vaccine described herein, to thereby inducing in the subject an immuneresponse specific to hMPV and/or hPIV3, wherein antibody titer ofantibodies against hMPV and/or hPIV3 in the subject is increasedfollowing vaccination relative to antibody titer in a subject vaccinatedwith a prophylactically effective dose of a traditional vaccine againsthMPV and/or hPIV3.

In some embodiments, a RNA (e.g., mRNA) vaccine (e.g., a hMPV/hPIV3 RNAvaccine) capable of eliciting an immune response is administeredintramuscularly via a composition including a compound according toFormula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) (e.g.,Compound 3, 18, 20, 25, 26, 29, 30, 60, 108-112, or 122).

A prophylactically effective dose is a therapeutically effective dosethat prevents infection with the virus at a clinically acceptable level.In some embodiments the therapeutically effective dose is a dose listedin a package insert for the vaccine. A traditional vaccine, as usedherein, refers to a vaccine other than the RNA (e.g., mRNA) vaccines ofthe present disclosure. For instance, a traditional vaccine includes butis not limited to live/attenuated microorganism vaccines,killed/inactivated microorganism vaccines, subunit vaccines, proteinantigen vaccines, DNA vaccines, VLP vaccines, etc. In exemplaryembodiments, a traditional vaccine is a vaccine that has achievedregulatory approval and/or is registered by a national drug regulatorybody, for example the Food and Drug Administration (FDA) in the UnitedStates or the European Medicines Agency (EMA).

In some embodiments the anti-hMPV F protein and/or anti-hPIV F proteinantibody titer in the subject is increased 1 log to 10 log followingvaccination relative to anti-hMPV F protein and/or anti-hPIV F proteinantibody titer in a subject vaccinated with a prophylactically effectivedose of a traditional vaccine against hMPV and/or hPIV3.

In some embodiments the anti-hMPV F protein and/or anti-hPIV F proteinantibody titer in the subject is increased 1 log, 2 log, 3 log, 5 log or10 log following vaccination relative to anti-hMPV F protein and/oranti-hPIV F protein antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against hMPVand/or hPIV3.

A method of eliciting an immune response in a subject against hMPVand/or hPIV3 is provided in other aspects of the disclosure. The methodinvolves administering to the subject an effective amount of a RNA(e.g., mRNA) vaccine described herein, to thereby inducing in thesubject an immune response specific to hMPV and/or hPIV3, wherein theimmune response in the subject is equivalent to an immune response in asubject vaccinated with a traditional vaccine against the hMPV and/orhPIV3 at 2 times to 100 times the dosage level relative to the RNA(e.g., mRNA) vaccine.

In some embodiments, the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at2, 3, 4, 5, 10, 50, 100 times the dosage level relative to the hMPVand/or hPIV3RNA (e.g., mRNA) vaccine. In some embodiments the immuneresponse in the subject is equivalent to an immune response in a subjectvaccinated with a traditional vaccine at 10-100 times, or 100-1000times, the dosage level relative to the hMPV and/or hPIV3RNA (e.g.,mRNA) vaccine. In some embodiments the immune response is assessed bydetermining [protein] antibody titer in the subject.

In some embodiments, the immune response in the subject is induced 2days earlier, or 3 days earlier, relative to an immune response inducedin a subject vaccinated with a prophylactically effective dose of atraditional vaccine. In some embodiments the immune response in thesubject is induced 1 week, 2 weeks, 3 weeks, 5 weeks, or 10 weeksearlier relative to an immune response induced in a subject vaccinatedwith a prophylactically effective dose of a traditional vaccine.

Therapeutic and Prophylactic Compositions

Provided herein are compositions (e.g., pharmaceutical compositions),methods, kits and reagents for prevention, treatment or diagnosis ofhMPV and/or hPIV3 in humans and other mammals, for example. The RNA(e.g. mRNA) vaccines can be used as therapeutic or prophylactic agents.They may be used in medicine to prevent and/or treat infectious disease.In some embodiments, the respiratory RNA (e.g., mRNA) vaccines of thepresent disclosure are used fin the priming of immune effector cells,for example, to activate peripheral blood mononuclear cells (PBMCs) exvivo, which are then infused (re-infused) into a subject.

In some embodiments, a RNA vaccine containing RNA (e.g., mRNA)polynucleotides as described herein can be administered to a subject(e.g., a mammalian subject, such as a human subject), and the RNA (e.g.,mRNA) polynucleotides are translated in vivo to produce hMPV and/orhPIV3 F protein.

hMPV/hPIV3RNA (e.g., mRNA) vaccines may be induced for translation of apolypeptide (e.g., antigen or immunogen) in a cell, tissue or organism.In some embodiments, such translation occurs in vivo, although suchtranslation may occur ex vivo, in culture or in vitro. In someembodiments, the cell, tissue or organism is contacted with an effectiveamount of a composition containing a RNA (e.g., mRNA) vaccine thatcontains a polynucleotide that has at least one a translatable regionencoding an antigenic polypeptide (e.g., hMPV F protein and/or hPIV3 Fprotein).

An “effective amount” of an RNA (e.g. mRNA) vaccine is provided based,at least in part, on the target tissue, target cell type, means ofadministration, physical characteristics of the polynucleotide (e.g.,size, and extent of modified nucleosides) and other components of thevaccine, and other determinants. In general, an effective amount of theRNA (e.g., mRNA) vaccine composition provides an induced or boostedimmune response as a function of antigen production in the cell,preferably more efficient than a composition containing a correspondingunmodified polynucleotide encoding the same antigen or a peptideantigen. Increased antigen production may be demonstrated by increasedcell transfection (the percentage of cells transfected with the RNA,e.g., mRNA, vaccine), increased protein translation from thepolynucleotide, decreased nucleic acid degradation (as demonstrated, forexample, by increased duration of protein translation from a modifiedpolynucleotide), or altered antigen specific immune response of the hostcell. In some embodiments, RNA (e.g. mRNA) vaccines (includingpolynucleotides their encoded polypeptides) in accordance with thepresent disclosure may be used for treatment of hMPV and/or hPIV3.

RNA (e.g. mRNA) vaccines may be administered prophylactically ortherapeutically as part of an active immunization scheme to healthyindividuals or early in infection during the incubation phase or duringactive infection after onset of symptoms.

In some embodiments, a subject is an elderly human subject (e.g., anindividual that is at least 65 years of age). For example, an elderlyhuman subject may be 65, 66, 67, 68, 70, 71,72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100 years of age or older.

In some embodiments, a subject is a child (e.g., an individual that is 5years of age or younger. For example, a subject that is a child may be5, 4, 3, 2, 1 years of age or younger. In some embodiments, a subjectthat is a child may be 6-12 months of age. For example, a subject thatis a child may be 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 7-8, 7-9, 7-10, 7-11,7-12, 8-9, 8-10, 8-11, 8-12, 9-10, 9-11, 9-12, 10-11, 10-12, or 11-12months of age. In some embodiments, a subject that is a child may be 6months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 monthsof age.

In some embodiments, the RNA (e.g. mRNA) vaccines described herein areused for preventing a lower respiratory hMPV and/or hPIV3 infection in asubject having a pulmonary condition. A “pulmonary condition,” as usedherein, encompasses any condition that is associated with or results inimpaired or reduce pulmonary function. In some embodiments, thepulmonary condition is associated with Chronic Obstructive PulmonaryDisease (COPD), asthma, congestive heart failure, diabetes, and anycombination thereof. In some embodiments, the respiratory RNA (e.g.mRNA) vaccines described herein are used for preventing a lowerrespiratory hMPV and/or hPIV3 infection in a immunocompromised subject(e.g., an AIDS patient or a transplant recipient).

In some embodiments, the amount of RNA (e.g., mRNA) vaccine of thepresent disclosure provided to a cell, a tissue or a subject may be anamount effective for immune prophylaxis.

RNA (e.g. mRNA) vaccines may be administrated with other prophylactic ortherapeutic compounds. As a non-limiting example, a prophylactic ortherapeutic compound may be an adjuvant or a booster. As used herein,when referring to a prophylactic composition, such as a vaccine, theterm “booster” refers to an extra administration of the prophylactic(vaccine) composition. A booster (or booster vaccine) may be given afteran earlier administration of the prophylactic composition. The time ofadministration between the initial administration of the prophylacticcomposition and the booster may be, but is not limited to, 1 minute, 2minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours,12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days,3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years,11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80years, 85 years, 90 years, 95 years or more than 99 years. In someembodiments, the time of administration between the initialadministration of the prophylactic composition and the booster may be,but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3months, 6 months or 1 year.

In some embodiments, hMPV/hPIV3 RNA (e.g. mRNA) vaccines may beadministered intramuscularly or intradermally, similarly to theadministration of inactivated vaccines known in the art.

RNA (e.g. mRNA) vaccines may be utilized in various settings dependingon the prevalence of the infection or the degree or level of unmetmedical need. As a non-limiting example, the RNA (e.g., mRNA) vaccinesmay be utilized to treat and/or prevent a variety of respiratoryinfections. RNA (e.g., mRNA) vaccines have superior properties in thatthey produce much larger antibody titers and produce responses earlythan commercially available anti-viral agents/compositions.

Provided herein are pharmaceutical compositions including RNA (e.g.mRNA) vaccines and RNA (e.g. mRNA) vaccine compositions and/or complexesoptionally in combination with one or more pharmaceutically acceptableexcipients.

RNA (e.g. mRNA) vaccines may be formulated or administered alone or inconjunction with one or more other components. For instance, hMPV/hPIV3RNA (e.g., mRNA) vaccines (vaccine compositions) may comprise othercomponents including, but not limited to, adjuvants.

In some embodiments, RNA (e.g. mRNA) vaccines do not include an adjuvant(they are adjuvant free).

RNA (e.g. mRNA) vaccines may be formulated or administered incombination with one or more pharmaceutically-acceptable excipients. Insome embodiments, vaccine compositions comprise at least one additionalactive substances, such as, for example, a therapeutically-activesubstance, a prophylactically-active substance, or a combination ofboth. Vaccine compositions may be sterile, pyrogen-free or both sterileand pyrogen-free. General considerations in the formulation and/ormanufacture of pharmaceutical agents, such as vaccine compositions, maybe found, for example, in Remington: The Science and Practice ofPharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporatedherein by reference in its entirety).

In some embodiments, hMPV/hPIV3 RNA (e.g. mRNA) vaccines areadministered to humans, human patients or subjects. For the purposes ofthe present disclosure, the phrase “active ingredient” generally refersto the RNA (e.g., mRNA) vaccines or the polynucleotides containedtherein, for example, RNA polynucleotides (e.g., mRNA polynucleotides)encoding antigenic polypeptides (e.g., F proteins).

Formulations of the RNA vaccine compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient (e.g., mRNA polynucleotide) intoassociation with an excipient and/or one or more other accessoryingredients, and then, if necessary and/or desirable, dividing, shapingand/or packaging the product into a desired single- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

RNA (e.g. mRNA) vaccines can be formulated using one or more excipientsto: (1) increase stability; (2) increase cell transfection; (3) permitthe sustained or delayed release (e.g., from a depot formulation); (4)alter the biodistribution (e.g., target to specific tissues or celltypes); (5) increase the translation of encoded protein in vivo; and/or(6) alter the release profile of encoded protein (antigen) in vivo. Inaddition to traditional excipients such as any and all solvents,dispersion media, diluents, or other liquid vehicles, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, excipients can include, withoutlimitation, lipidoids, liposomes, lipid nanoparticles, polymers,lipoplexes, core-shell nanoparticles, peptides, proteins, cellstransfected with RNA (e.g. mRNA)vaccines (e.g., for transplantation intoa subject), hyaluronidase, nanoparticle mimics and combinations thereof.

Modes of Vaccine Administration

The hMPV/hPIV3 RNA (e.g. mRNA) vaccines described herein may beadministered by any route which results in a therapeutically effectiveoutcome. These include, but are not limited, to intradermal,intramuscular, and/or subcutaneous administration. A RNA (e.g. mRNA)vaccine pharmaceutical composition described herein can be formulatedinto a dosage form described herein, such as an intranasal,intratracheal, or injectable (e.g., intravenous, intraocular,intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal,and subcutaneous).

The present disclosure provides methods comprising administering RNA(e.g., mRNA) vaccines to a subject in need thereof. The exact amountrequired will vary from subject to subject, depending on the species,age, and general condition of the subject, the severity of the disease,the particular composition, its mode of administration, its mode ofactivity, and the like. RNA (e.g., mRNA) vaccines compositions aretypically formulated in dosage unit form for ease of administration anduniformity of dosage. It will be understood, however, that the totaldaily usage of RNA (e.g., mRNA) vaccine compositions may be decided bythe attending physician within the scope of sound medical judgment. Thespecific therapeutically effective, prophylactically effective, orappropriate imaging dose level for any particular patient will dependupon a variety of factors including the disorder being treated and theseverity of the disorder; the activity of the specific compoundemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment; drugs used in combination orcoincidental with the specific compound employed; and like factors wellknown in the medical arts.

In some embodiments, RNA (e.g. mRNA) vaccine compositions may beadministered at dosage levels sufficient to deliver 0.0001 mg/kg to 100mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005 mg/kg to 0.05 mg/kg, 0.001 mg/kgto 0.005 mg/kg, 0.05 mg/kg to 0.5 mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1mg/kg to 40 mg/kg, 0.5 mg/kg to 30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1mg/kg to 10 mg/kg, or 1 mg/kg to 25 mg/kg, of subject body weight perday, one or more times a day, per week, per month, etc. to obtain thedesired therapeutic, diagnostic, prophylactic, or imaging effect (see,e.g., the range of unit doses described in International Publication NoWO2013078199, the contents of which are herein incorporated by referencein their entirety). The desired dosage may be delivered three times aday, two times a day, once a day, every other day, every third day,every week, every two weeks, every three weeks, every four weeks, every2 months, every three months, every 6 months, etc. In some embodiments,the desired dosage may be delivered using multiple administrations(e.g., two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, or more administrations). When multipleadministrations are employed, split dosing regimens such as thosedescribed herein may be used. In exemplary embodiments, RNA (e.g., mRNA)vaccines compositions may be administered at dosage levels sufficient todeliver 0.0005 mg/kg to 0.01 mg/kg, e.g., about 0.0005 mg/kg to about0.0075 mg/kg, e.g., about 0.0005 mg/kg, about 0.001 mg/kg, about 0.002mg/kg, about 0.003 mg/kg, about 0.004 mg/kg or about 0.005 mg/kg.

In some embodiments, RNA (e.g., mRNA) vaccine compositions may beadministered once or twice (or more) at dosage levels sufficient todeliver 0.025 mg/kg to 0.250 mg/kg, 0.025 mg/kg to 0.500 mg/kg, 0.025mg/kg to 0.750 mg/kg, or 0.025 mg/kg to 1.0 mg/kg.

In some embodiments, RNA (e.g., mRNA) vaccine compositions may beadministered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 andDay 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 andDay 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later,Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12 monthslater, Day 0 and 18 months later, Day 0 and 2 years later, Day 0 and 5years later, or Day 0 and 10 years later) at a total dose of or atdosage levels sufficient to deliver a total dose of 0.0100 mg, 0.025 mg,0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg,0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg,0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg,0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg,0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg,0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher and lower dosages andfrequency of administration are encompassed by the present disclosure.For example, a RNA (e.g., mRNA) vaccine composition may be administeredthree or four times.

In some embodiments, hMPV/hPIV3 RNA (e.g., mRNA) vaccine compositionsmay be administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 monthslater, Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12months later, Day 0 and 18 months later, Day 0 and 2 years later, Day 0and 5 years later, or Day 0 and 10 years later) at a total dose of or atdosage levels sufficient to deliver a total dose of 0.010 mg, 0.025 mg,0.100 mg or 0.400 mg.

In some embodiments, the hMPV/hPIV3 RNA (e.g., mRNA) vaccine for use ina method of vaccinating a subject is administered to the subject as asingle dosage of between 10 μg/kg and 400 μg/kg of the nucleic acidvaccine (in an effective amount to vaccinate the subject). In someembodiments the RNA (e.g., mRNA) vaccine for use in a method ofvaccinating a subject is administered to the subject as a single dosageof between 10 μg and 400 μg of the nucleic acid vaccine (in an effectiveamount to vaccinate the subject).

In some embodiments, a hMPV/hPIV3 RNA (e.g., mRNA) vaccine for use in amethod of vaccinating a subject is administered to the subject as asingle dosage of 2-1000 μg (e.g., a single dosage of mRNA encoding hMPV,hPIV3, and/or RSV antigen). In some embodiments, a RNA (e.g., mRNA)vaccine for use in a method of vaccinating a subject is administered tothe subject as a single dosage of 5-100 μg (e.g., a single dosage ofmRNA encoding hMPV and/or hPIV3 F protein). In some embodiments, a RNA(e.g., mRNA) vaccine is administered to the subject as a single dosageof 2, 5, 10, 15, 20 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 μg. For example, aRNA (e.g., mRNA) vaccine may be administered to a subject as a singledose of 5-100, 10-100, 15-100, 20-100, 25-100, 25-500, 50-100, 50-500,50-1000, 100-500, 100-1000, 250-500, 250-1000, or 500-1000 μg. In someembodiments, a RNA (e.g., mRNA) vaccine for use in a method ofvaccinating a subject is administered to the subject as two dosages, thecombination of which equals 10-1000 μg of the RNA (e.g., mRNA) vaccine.

hMPV/hPIV3 RNA (e.g., mRNA) Vaccine Formulations and Methods of Use

Some aspects of the present disclosure provide formulations of ahMPV/hPIV3 RNA (e.g., mRNA) vaccine, wherein the RNA (e.g., mRNA)vaccine is formulated in an effective amount to produce an antigenspecific immune response in a subject (e.g., production of antibodiesspecific to an hMPV and/or hPIV3 F protein). “An effective amount” is adose of an RNA (e.g., mRNA) vaccine effective to produce anantigen-specific immune response (e.g., that results in an ability toclear the virus more rapidly and/or a reduction in infectious virus inthe nasal and/or lung passages upon exposure to the virus). Alsoprovided herein are methods of inducing an antigen-specific immuneresponse in a subject.

In some embodiments, the antigen-specific immune response ischaracterized by measuring an anti-hMPV and/or anti-PIV3 F proteinantibody titer produced in a subject administered a RNA (e.g., mRNA)vaccine as provided herein. An antibody titer is a measurement of theamount of antibodies within a subject, for example, antibodies that arespecific to a particular antigen (e.g., an anti-hMPV and/or anti-PIV3 Fprotein) or epitope of an antigen. Antibody titer is typically expressedas the inverse of the greatest dilution that provides a positive result.Enzyme-linked immunosorbent assay (ELISA) is a common assay fordetermining antibody titers, for example.

In some embodiments, an antibody titer is used to assess whether asubject has had an infection or to determine whether immunizations arerequired. In some embodiments, an antibody titer is used to determinethe strength of an autoimmune response, to determine whether a boosterimmunization is needed, to determine whether a previous vaccine waseffective, and to identify any recent or prior infections. In accordancewith the present disclosure, an antibody titer may be used to determinethe strength of an immune response induced in a subject by the RNA(e.g., mRNA) vaccine.

In some embodiments, an antibody induced by a RNA (e.g., mRNA) vaccineis a neutralizing antibody against the hMPV and/or hPIV3. A neutralizingtiter is produced by neutralizing antibody against hMPV/PIV3 F proteinas measured in serum of the subject. In some embodiments, an effectivedose of the hMPV/PIV3 RNA (e.g., mRNA) vaccine is sufficient to producemore than a 500 neutralization titer. For example, an effective dose ofthe hMPV/PIV3 RNA (e.g., mRNA) vaccine is sufficient to produce a1000-10,000 neutralization titer. In some embodiments, an effective doseof the hMPV/PIV3 RNA (e.g., mRNA) vaccine is sufficient to produce a1000-2000, 1000-3000, 1000-4000, 1000-5000, 1000-6000, 1000-7000,1000-8000, 1000-9000, 1000-10,000, 2000-3000, 2000-4000, 2000-5000,2000-6000, 2000-7000, 2000-8000, 2000-9000, 2000-10,000, 3000-4000,3000-5000, 3000-6000, 3000-7000, 3000-8000, 3000-9000, 3000-10,000,4000-5000, 4000-6000, 4000-7000, 4000-8000, 4000-9000, 4000-10,000,5000-6000, 5000-7000, 5000-8000, 5000-9000; 5000-10,000, 6000-7000,6000-8000, 6000-9000, 6000-10,000, 7000-8000, 7000-9000, 7000-10,000,8000-9000, 8000-10,000, or a 9000-10,000 neutralization titer. In someembodiments, an effective dose of the hMPV/PIV3 RNA (e.g., mRNA) vaccineis sufficient to produce a 500, 1000, 1500, 2000, 2500, 3000, 3500,4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500,10,000, 11000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000,19000, 20,000 or higher neutralizing titer. In some embodiments,neutralizing titer is produced 1-72 hours post administration. Forexample, neutralizing titers may be produced 1-10, 1-20, 1-30, 1-40,1-50, 1-60, 1-70, 1-72, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-72,20-30, 20-40, 20-50, 20-60, 20-70, 20-72, 30-40, 30-50, 30-60, 30-70,30-72, 40-50, 40-60, 40-70, 40-72, 50-60, 50-70, 50-72, 60-70, 60-72, or70-72 hours post administration. In some embodiments, neutralizingtiters may be produced 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 56, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, or 72 hours post administration. In some embodiments,neutralizing titer is produced within 14 days of vaccine administration.In some embodiments, neutralizing titer is produced within 5, 6, 7, 8,9, 10, 11, 12, 13, or 14 days of vaccine administration.

In some embodiments, an effective dose of the hMPV/PIV3 RNA (e.g., mRNA)vaccine is comparable to the dose of the vaccine required to produce, ina cotton rat model or an African Green Monkey model, a neutralizationtiter of at least 6, at least 7, at least 8, or at least 9 on a log 2based scale, as measured by 60% plaque reduction neutralization test(PRNT). In some embodiments, an effective dose of the hMPV/PIV3 RNA(e.g., mRNA) vaccine is comparable to the dose of the vaccine requiredto produce, in a cotton rat model or an African Green Monkey model, aneutralization titer of 6, 7, 8, 9, 10, 11 or 12 on a log 2 based scale,as measured by 60% plaque reduction neutralization test (PRNT). In someembodiments, an effective dose of the hMPV/PIV3 RNA (e.g., mRNA) vaccineis comparable to the dose of the vaccine required to produce, in acotton rat model or an African Green Monkey model, a neutralizationtiter of 6-12, 7-12, 8-12, or 9-12 on a log 2 based scale, as measuredby 60% plaque reduction neutralization test (PRNT). In some embodiments,the high neutralizing antibody titer is induced within 14 days ofvaccine administration. In some embodiments, the high neutralizingantibody titer is induced within 21 days of vaccine administration. Insome embodiments, the high neutralizing antibody titer is induced within28 days of vaccine administration.

In some embodiments, an anti-hMPV F protein and/or anti-PIV3 F proteinantibody titer produced in a subject is increased by at least 1 logrelative to a control. For example, anti-hMPV F protein and/or anti-PIV3F protein antibody titer produced in a subject may be increased by atleast 1.5, at least 2, at least 2.5, or at least 3 log relative to acontrol. In some embodiments, the anti-hMPV F protein and/or anti-PIV3 Fprotein polypeptide antibody titer produced in the subject is increasedby 1, 1.5, 2, 2.5 or 3 log relative to a control. In some embodiments,the anti-hMPV F protein and/or anti-PIV3 F protein antibody titerproduced in the subject is increased by 1-3 log relative to a control.For example, the anti-hMPV F protein and/or anti-PIV3 F protein antibodytiter produced in a subject may be increased by 1-1.5, 1-2, 1-2.5, 1-3,1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3, or 2.5-3 log relative to a control.

In some embodiments, the anti-hMPV F protein and/or anti-PIV3 F proteinantibody titer produced in a subject is increased at least 2 timesrelative to a control. For example, the anti-hMPV F protein and/oranti-PIV3 F protein antibody titer produced in a subject may beincreased at least 3 times, at least 4 times, at least 5 times, at least6 times, at least 7 times, at least 8 times, at least 9 times, or atleast 10 times relative to a control. In some embodiments, the anti-hMPVF protein and/or anti-PIV3 F protein antibody titer produced in thesubject is increased 2, 3, 4, 5, 6, 7, 8, 9, or 10 times relative to acontrol. In some embodiments, the anti-hMPV F protein and/or anti-PIV3 Fprotein antibody titer produced in a subject is increased 2-10 timesrelative to a control. For example, the anti-hMPV F protein and/oranti-PIV3 F protein antibody titer produced in a subject may beincreased 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7,3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6,6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 times relativeto a control.

A control, in some embodiments, is the anti-hMPV F protein and/oranti-PIV3 F protein antibody titer produced in a subject who has notbeen administered a RNA (e.g., mRNA) vaccine of the present disclosure.In some embodiments, a control is an anti-hMPV F protein and/oranti-PIV3 F protein (antibody titer produced in a subject who has beenadministered a live attenuated hMPV and/or hPIV3 vaccine or aninactivated hMPV and/or hPIV3 vaccine. An attenuated vaccine is avaccine produced by reducing the virulence of a viable (live). Anattenuated virus is altered in a manner that renders it harmless or lessvirulent relative to live, unmodified virus. In some embodiments, acontrol is an anti-hMPV F protein and/or anti-PIV3 F protein antibodytiter produced in a subject administered inactivated hMPV and/or hPIV3vaccine. In some embodiments, a control is anti-hMPV F protein and/oranti-PIV3 F protein antibody titer produced in a subject administered arecombinant or purified hMPV and/or hPIV3 protein vaccine. Recombinantprotein vaccines typically include protein antigens that either havebeen produced in a heterologous expression system (e.g., bacteria oryeast) or purified from large amounts of the pathogenic organism. Insome embodiments, a control is an antibody titer produced in a subjectwho has been administered an hMPV and/or hPIV3 virus-like particle (VLP)vaccine. For example, an hMPV VLP vaccine used as a control may be ahMPV VLPs, comprising (or consisting of) viral matrix (M) and fusion (F)proteins, generated by expressing viral proteins in suspension-adaptedhuman embryonic kidney epithelial (293-F) cells (see, e.g., Cox R G etal., J Virol. 2014 June; 88(11): 6368-6379, the contents of which areherein incorporated by reference).

In some embodiments, an effective amount of a hMPV/hPIV3 RNA (e.g.,mRNA) vaccine is a dose that is reduced compared to the standard of caredose of a recombinant hMPV and/or hPIV3 protein vaccine. A “standard ofcare,” as provided herein, refers to a medical or psychologicaltreatment guideline and can be general or specific. “Standard of care”specifies appropriate treatment based on scientific evidence andcollaboration between medical professionals involved in the treatment ofa given condition. It is the diagnostic and treatment process that aphysician/clinician should follow for a certain type of patient, illnessor clinical circumstance. A “standard of care dose,” as provided herein,refers to the dose of a recombinant or purified hMPV and/or hPIV3protein vaccine, or a live attenuated or inactivated hMPV and/or hPIV3vaccine, that a physician/clinician or other medical professional wouldadminister to a subject to treat or prevent hMPV and/or hPIV3, or ahMPV- and/or hPIV3-related condition, while following the standard ofcare guideline for treating or preventing hMPV and/or hPIV3, or a hMPV-and/or hPIV3-related condition.

In some embodiments, the an anti-hMPV F protein and/or anti-PIV3 Fprotein antibody titer produced in a subject administered an effectiveamount of a RNA (e.g., mRNA) vaccine is equivalent to an an anti-hMPV Fprotein and/or anti-PIV3 F protein antibody titer produced in a controlsubject administered a standard of care dose of a recombinant orpurified hMPV and/or hPIV3 protein vaccine or a live attenuated orinactivated hMPV and/or hPIV3 vaccine.

In some embodiments, an effective amount of a RNA (e.g., mRNA) vaccineis a dose equivalent to an at least 2-fold reduction in a standard ofcare dose of a recombinant or purified hMPV and/or hPIV3 proteinvaccine. For example, an effective amount of a RNA (e.g., mRNA) vaccinemay be a dose equivalent to an at least 3-fold, at least 4-fold, atleast 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, atleast 9-fold, or at least 10-fold reduction in a standard of care doseof a recombinant or purified hMPV and/or hPIV3 protein vaccine. In someembodiments, an effective amount of a RNA (e.g., mRNA) vaccine is a doseequivalent to an at least at least 100-fold, at least 500-fold, or atleast 1000-fold reduction in a standard of care dose of a recombinant orpurified hMPV and/or hPIV3 protein vaccine. In some embodiments, aneffective amount of a RNA (e.g., mRNA) vaccine is a dose equivalent to a2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 20-, 50-, 100-, 250-, 500-, or1000-fold reduction in a standard of care dose of a recombinant orpurified hMPV and/or hPIV3 protein vaccine. In some embodiments, theanti-hMPV F protein and/or anti-hPIV F protein antibody titer producedin a subject administered an effective amount of a RNA (e.g., mRNA)vaccine is equivalent to an anti-hMPV F protein and/or anti-hPIV Fprotein antibody titer produced in a control subject administered thestandard of care dose of a recombinant or protein hMPV and/or hPIV3,protein vaccine or a live attenuated or inactivated hMPV and/or hPIV3vaccine. In some embodiments, an effective amount of a RNA (e.g., mRNA)vaccine is a dose equivalent to a 2-fold to 1000-fold (e.g., 2-fold to100-fold, 10-fold to 1000-fold) reduction in the standard of care doseof a recombinant or purified hMPV and/or hPIV3 protein vaccine, whereinthe anti-hMPV F protein and/or anti-hPIV F protein antibody titerproduced in the subject is equivalent to an anti-hMPV F protein and/oranti-hPIV F protein antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified hMPVand/or hPIV3 protein vaccine or a live attenuated or inactivated hMPVand/or hPIV3 vaccine.

In some embodiments, the effective amount of a hMPV/hPIV3 RNA (e.g.,mRNA) vaccine is a dose equivalent to a 2 to 1000-, 2 to 900-, 2 to800-, 2 to 700-, 2 to 600-, 2 to 500-, 2 to 400-, 2 to 300-, 2 to 200-,2 to 100-, 2 to 90-, 2 to 80-, 2 to 70-, 2 to 60-, 2 to 50-, 2 to 40-, 2to 30-, 2 to 20-, 2 to 10-, 2 to 9-, 2 to 8-, 2 to 7-, 2 to 6-, 2 to 5-,2 to 4-, 2 to 3-, 3 to 1000-, 3 to 900-, 3 to 800-, 3 to 700-, 3 to600-, 3 to 500-, 3 to 400-, 3 to 3 to 00-, 3 to 200-, 3 to 100-, 3 to90-, 3 to 80-, 3 to 70-, 3 to 60-, 3 to 50-, 3 to 40-, 3 to 30-, 3 to20-, 3 to 10-, 3 to 9-, 3 to 8-, 3 to 7-, 3 to 6-, 3 to 5-, 3 to 4-, 4to 1000-, 4 to 900-, 4 to 800-, 4 to 700-, 4 to 600- , 4 to 500-, 4 to400-, 4 to 4 to 00-, 4 to 200-, 4 to 100-, 4 to 90-, 4 to 80-, 4 to 70-,4 to 60-, 4 to 50-, 4 to 40-, 4 to 30-, 4 to 20-, 4 to 10-, 4 to 9-, 4to 8-, 4 to 7-, 4 to 6-, 4 to 5-, 4 to 4-, 5 to 1000-, 5 to 900-, 5 to800-, 5 to 700-, 5 to 600-, 5 to 500-, 5 to 400-, 5 to 300-, 5 to 200-,5 to 100-, 5 to 90-, 5 to 80-, 5 to 70-, 5 to 60-, 5 to 50-, 5 to 40-, 5to 30-, 5 to 20-, 5 to 10-, 5 to 9- , 5 to 8-, 5 to 7-, 5 to 6-, 6 to1000-, 6 to 900-, 6 to 800-, 6 to 700-, 6 to 600-, 6 to 500-, 6 to 400-,6 to 300-, 6 to 200-, 6 to 100-, 6 to 90-, 6 to 80-, 6 to 70-, 6 to 60-,6 to 50-, 6 to 40-, 6 to 30-, 6 to 20-, 6 to 10-, 6 to 9-, 6 to 8-, 6 to7-, 7 to 1000-, 7 to 900-, 7 to 800-, 7 to 700-, 7 to 600-, 7 to 500-, 7to 400-, 7 to 300-, 7 to 200-, 7 to 100-, 7 to 90-, 7 to 80-, 7 to 70-,7 to 60-, 7 to 50-, 7 to 40-, 7 to 30-, 7 to 20-, 7 to 10-, 7 to 9-, 7to 8-, 8 to 1000-, 8 to 900-, 8 to 800-, 8 to 700-, 8 to 600-, 8 to500-, 8 to 400-, 8 to 300-, 8 to 200-, 8 to 100-, 8 to 90-, 8 to 80-, 8to 70-, 8 to 60-, 8 to 50-, 8 to 40-, 8 to 30-, 8 to 20-, 8 to 10-, 8 to9-, 9 to 1000-, 9 to 900-, 9 to 800-, 9 to 700-, 9 to 600-, 9 to 500-, 9to 400-, 9 to 300-, 9 to 200-, 9 to 100-, 9 to 90-, 9 to 80-, 9 to 70-,9 to 60-, 9 to 50-, 9 to 40-, 9 to 30-, 9 to 20-, 9 to 10-, 10 to 1000-,10 to 900-, 10 to 800-, 10 to 700-, 10 to 600-, 10 to 500-, 10 to 400-,10 to 300-, 10 to 200-, 10 to 100-, 10 to 90-, 10 to 80-, 10 to 70-, 10to 60-, 10 to 50-, 10 to 40-, 10 to 30-, 10 to 20-, 20 to 1000-, 20 to900-, 20 to 800-, 20 to 700-, 20 to 600-, 20 to 500-, 20 to 400-, 20 to300-, 20 to 200-, 20 to 100-, 20 to 90-, 20 to 80-, 20 to 70-, 20 to60-, 20 to 50-, 20 to 40-, 20 to 30-, 30 to 1000-, 30 to 900-, 30 to800-, 30 to 700-, 30 to 600-, 30 to 500-, 30 to 400-, 30 to 300-, 30 to200-, 30 to 100-, 30 to 90-, 30 to 80-, 30 to 70-, 30 to 60-, 30 to 50-,30 to 40-, 40 to 1000-, 40 to 900-, 40 to 800-, 40 to 700-, 40 to 600-,40 to 500-, 40 to 400-, 40 to 300-, 40 to 200-, 40 to 100-, 40 to 90-,40 to 80-, 40 to 70-, 40 to 60-, 40 to 50-, 50 to 1000-, 50 to 900-, 50to 800-, 50 to 700-, 50 to 600-, 50 to 500-, 50 to 400-, 50 to 300-, 50to 200-, 50 to 100-, 50 to 90-, 50 to 80-, 50 to 70-, 50 to 60-, 60 to1000-, 60 to 900-, 60 to 800-, 60 to 700-, 60 to 600-, 60 to 500-, 60 to400-, 60 to 300-, 60 to 200-, 60 to 100-, 60 to 90-, 60 to 80-, 60 to70-, 70 to 1000-, 70 to 900-, 70 to 800-, 70 to 700-, 70 to 600-, 70 to500-, 70 to 400-, 70 to 300-, 70 to 200-, 70 to 100-, 70 to 90-, 70 to80-, 80 to 1000-, 80 to 900-, 80 to 800-, 80 to 700-, 80 to 600-, 80 to500-, 80 to 400-, 80 to 300-, 80 to 200-, 80 to 100-, 80 to 90-, 90 to1000-, 90 to 900-, 90 to 800-, 90 to 700-, 90 to 600-, 90 to 500-, 90 to400-, 90 to 300-, 90 to 200-, 90 to 100-, 100 to 1000-, 100 to 900-, 100to 800-, 100 to 700-, 100 to 600-, 100 to 500-, 100 to 400-, 100 to300-, 100 to 200-, 200 to 1000-, 200 to 900-, 200 to 800-, 200 to 700-,200 to 600-, 200 to 500-, 200 to 400-, 200 to 300-, 300 to 1000-, 300 to900-, 300 to 800-, 300 to 700-, 300 to 600-, 300 to 500-, 300 to 400-,400 to 1000-, 400 to 900-, 400 to 800-, 400 to 700-, 400 to 600-, 400 to500-, 500 to 1000-, 500 to 900-, 500 to 800-, 500 to 700-, 500 to 600-,600 to 1000-, 600 to 900-, 600 to 800-, 600 to 700-, 700 to 1000-, 700to 900-, 700 to 800-, 800 to 1000-, 800 to 900-, or 900 to 1000-foldreduction in the standard of care dose of a recombinant hMPV and/orhPIV3 protein vaccine. In some embodiments, the anti-HMP F proteinand/or anti-hPIV3 F protein antibody titer produced in the subject isequivalent to an anti-hMPV F protein and/or anti-hPIV F protein antibodytiter produced in a control subject administered the standard of caredose of a recombinant or purified hMPV and/or hPIV3 protein vaccine or alive attenuated or inactivated hMPV and/or hPIV3vaccine. In someembodiments, the effective amount is a dose equivalent to (or equivalentto an at least) 2-, 3-, 4-,5-,6-, 7-, 8-, 9-, 10-, 20-, 30-, 40-, 50-,60-, 70-, 80-, 90-, 100-, 110-, 120-, 130-, 140-, 150-, 160-, 170-,1280-, 190-, 200-, 210-, 220-, 230-, 240-, 250-, 260-, 270-, 280-, 290-,300-, 310-, 320-, 330-, 340-, 350-, 360-, 370-, 380-, 390-, 400-, 410-,420-, 430-, 440-, 450-, 4360-, 470-, 480-, 490-, 500-, 510-, 520-, 530-,540-, 550-, 560-, 5760-, 580-, 590-, 600-, 610-, 620-, 630-, 640-, 650-,660-, 670-, 680-, 690-, 700-, 710-, 720-, 730-, 740-, 750-, 760-, 770-,780-, 790-, 800-, 810-, 820--, 830-, 840-, 850-, 860-, 870-, 880-, 890-,900-, 910-, 920-, 930-, 940-, 950-, 960-, 970-, 980-, 990-, or 1000-foldreduction in the standard of care dose of a recombinant hMPV and/orhPIV3 protein vaccine. In some embodiments, an anti-hMPV F proteinand/or anti-hPIV3 F protein antibody titer produced in the subject isequivalent to an an anti-hMPV F protein and/or anti-hPIV3 F proteinantibody titer produced in a control subject administered the standardof care dose of a recombinant or purified hMPV and/or hPIV3proteinvaccine or a live attenuated or inactivated hMPV and/or hPIV3 vaccine.

In some embodiments, the effective amount is 5 μg-100 μg of the RNApolynucleotide encoding hMPV F protein and/or 5 μg-100 μg of the RNApolynucleotide encoding hPIV3 F protein. For example, the effectiveamount may be 5 μg-10 μg, 5 μg-20 μg, 5 μg-30 μg, 5 μg-40 μg, 5 μg-50μg, 5 μg-60 μg, 5 μg-70 μg, 5 μg-80 μg, 5 μg-90 μg, 5 μg-100 μg, 10μg-20 μg, 10 μg-30 μg, 10 μg-40 μg, 10 μg-50 μg, 10 μg-60 μg, 10 μg-70μg, 10 μg-80 μg, 10 μg-90 μg, 10 μg-100 μg, 25 μg-30 μg, 25 μg-40 μg, 25μg-50 μg, 25 μg-60 μg, 25 μg-70 μg, 25 μg-80 μg, 25 μg-90 μg, 25 μg-100μg, 50 μg-60 μg, 50 μg-70 μg, 50 μg-80 μg, 50 μg-90 μg, or 50 μg-100 μgof the RNA polynucleotide encoding hMPV F protein and/or 5 μg-10 μg, 5μg-20 μg, 5 μg-30 μg, 5 μg-40 μg, 5 μg-50 μg, 5 μg-60 μg, 5 μg-70 μg, 5μg-80 μg, 5 μg-90 μg, 5 μg-100 μg, 10 μg-20 μg, 10 μg-30 μg, 10 μg-40μg, 10 μg-50 μg, 10 μg-60 μg, 10 μg-70 μg, 10 μg-80 μg, 10 μg-90 μg, 10μg-100 μg, 25 μg-30 μg, 25 μg-40 μg, 25 μg-50 μg, 25 μg-60 μg, 25 μg-70μg, 25 μg-80 μg, 25 μg-90 μg, 25 μg-100 μg, 50 μg-60 μg, 50 μg-70 μg, 50μg-80 μg, 50 μg-90 μg, or 50 μg-100 μg of the RNA polynucleotideencoding hPIV3 F protein. In some embodiments, the effective amount is 5μg, 10 μg, 12.5 μg, 20 μg, 25 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80μg, 90 μg, 100 μg of the RNA polynucleotide encoding hMPV F proteinand/or 5 μg, 10 μg, 12.5 μg, 20 μg, 25 μg, 30 μg, 40 μg, 50 μg, 60 μg,70 μg, 80 μg, 90 μg, 100 μg of the RNA polynucleotide encoding hPIV3 Fprotein. In some embodiments, the effective amount is 12.5 μg of the RNApolynucleotide encoding hMPV F protein and/or 12.5 μg of the RNApolynucleotide encoding hPIV3 F protein. In some embodiments, theeffective amount is 25 μg of the RNA polynucleotide encoding hMPV Fprotein and/or 25 μg of the RNA polynucleotide encoding hPIV3 F protein.In some embodiments, the effective amount is 50 μg of the RNApolynucleotide encoding hMPV F protein and/or 50 μg of the RNApolynucleotide encoding hPIV3 F protein. In some embodiments, theeffective amount is 100 μg of the RNA polynucleotide encoding hMPV Fprotein and/or 100 μg of the RNA polynucleotide encoding hPIV3 Fprotein.

In some embodiments, an effective dose of the RNA (e.g., mRNA) vaccinedescribed herein is sufficient to produce detectable levels of F proteinas measured in serum of the subject at 1-72 hours post administration.For example, hMPV F protein and/or hPIV3 F protein may be detected at 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, or 72 hours post administration. In some embodiments, an effectivedose of the RNA (e.g., mRNA) vaccine described herein is sufficient toproduce detectable levels of F protein as measured in serum of thesubject within 14 days of administration. In some embodiments, the cutoff index of the antigen is 1-2 (e.g., 1, 1.5, or 2). In someembodiments, wherein the effective dose is sufficient to produce a1,000-10,000 neutralization titer produced by neutralizing antibodyagainst hMPV F protein and/or hPIV3 F protein as measured in serum ofthe subject at 1-72 hours post administration. In some embodiments,wherein the effective dose is sufficient to produce a 1,000-10,000neutralization titer produced by neutralizing antibody against hMPV Fprotein and/or hPIV3 F protein as measured in serum of the subjectwithin 14 days of administration. In some embodiments, the cut-off indexof hMPV F protein and/or hPIV3 F protein is 1-2.

Vaccine Efficacy

Some aspects of the present disclosure provide formulations of thehMPV/hPIV3 RNA (e.g., mRNA) vaccine, wherein the hMPV/hPIV3 RNA vaccineis formulated in an effective amount to produce an antigen specificimmune response in a subject (e.g., production of antibodies specific toan anti-hMPV/hPIV3 antigen). “An effective amount” is a dose of ahMPV/hPIV3 RNA (e.g., mRNA) vaccine effective to produce anantigen-specific immune response. Also provided herein are methods ofinducing an antigen-specific immune response in a subject.

As used herein, an immune response to a vaccine or LNP of the presentdisclosure is the development in a subject of a humoral and/or acellular immune response to a (one or more) hMPV/hPIV3 protein(s)present in the vaccine. For purposes of the present disclosure, a“humoral” immune response refers to an immune response mediated byantibody molecules, including, e.g., secretory (IgA) or IgG molecules,while a “cellular” immune response is one mediated by T-lymphocytes(e.g., CD4+ helper and/or CD8+ T cells (e.g., CTLs) and/or other whiteblood cells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (CTLs). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote thedestruction of intracellular microbes or the lysis of cells infectedwith such microbes. Another aspect of cellular immunity involves andantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A cellular immune response also leads to theproduction of cytokines, chemokines, and other such molecules producedby activated T-cells and/or other white blood cells including thosederived from CD4+ and CD8+ T-cells.

In some embodiments, the antigen-specific immune response ischaracterized by measuring an anti-hMPV/hPIV3 antigen antibody titerproduced in a subject administered a hMPV/hPIV3 RNA (e.g., mRNA) vaccineas provided herein. An antibody titer is a measurement of the amount ofantibodies within a subject, for example, antibodies that are specificto a particular antigen (e.g., an anti-hMPV/hPIV3 antigen) or epitope ofan antigen. Antibody titer is typically expressed as the inverse of thegreatest dilution that provides a positive result. Enzyme-linkedimmunosorbent assay (ELISA) is a common assay for determining antibodytiters, for example.

In some embodiments, an antibody titer is used to assess whether asubject has had an infection or to determine whether immunizations arerequired. In some embodiments, an antibody titer is used to determinethe strength of an autoimmune response, to determine whether a boosterimmunization is needed, to determine whether a previous vaccine waseffective, and to identify any recent or prior infections. In accordancewith the present disclosure, an antibody titer may be used to determinethe strength of an immune response induced in a subject by thehMPV/hPIV3 RNA (e.g., mRNA) vaccine.

In some embodiments, an anti-hMPV/hPIV3 antigen antibody titer producedin a subject is increased by at least 1 log relative to a control. Forexample, anti-hMPV/hPIV3 antigen antibody titer produced in a subjectmay be increased by at least 1.5, at least 2, at least 2.5, or at least3 log relative to a control. In some embodiments, the anti-hMPV/hPIV3antigen antibody titer produced in the subject is increased by 1, 1.5,2, 2.5 or 3 log relative to a control. In some embodiments, theanti-hMPV/hPIV3 antigen antibody titer produced in the subject isincreased by 1-3 log relative to a control. For example, theanti-hMPV/hPIV3 antigen antibody titer produced in a subject may beincreased by 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3,or 2.5-3 log relative to a control.

In some embodiments, the anti-hMPV/hPIV3 antigen antibody titer producedin a subject is increased at least 2 times relative to a control. Forexample, the anti-hMPV/hPIV3 antigen antibody titer produced in asubject may be increased at least 3 times, at least 4 times, at least 5times, at least 6 times, at least 7 times, at least 8 times, at least 9times, or at least 10 times relative to a control. In some embodiments,the anti-hMPV/hPIV3 antigen antibody titer produced in the subject isincreased 2, 3, 4, 5, 6, 7, 8, 9, or 10 times relative to a control. Insome embodiments, the anti-hMPV/hPIV3 antigen antibody titer produced ina subject is increased 2-10 times relative to a control. For example,the anti-hMPV/hPIV3 antigen antibody titer produced in a subject may beincreased 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7,3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6,6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 times relativeto a control.

A control, in some embodiments, is the anti-hMPV/hPIV3 antigen antibodytiter produced in a subject who has not been administered a hMPV/hPIV3RNA (e.g., mRNA) vaccine. In some embodiments, a control is ananti-hMPV/hPIV3 antigen antibody titer produced in a subjectadministered a recombinant or purified hMPV/hPIV3 protein vaccine.Recombinant protein vaccines typically include protein antigens thateither have been produced in a heterologous expression system (e.g.,bacteria or yeast) or purified from large amounts of the pathogenicorganism.

In some embodiments, the ability of a hMPV/hPIV3 vaccine to be effectiveis measured in a murine model. For example, the hMPV/hPIV3 vaccines maybe administered to a murine model and the murine model assayed forinduction of neutralizing antibody titers. Viral challenge studies mayalso be used to assess the efficacy of a vaccine of the presentdisclosure. For example, the hMPV/hPIV3 vaccines may be administered toa murine model, the murine model challenged with hMPV/hPIV3, and themurine model assayed for survival and/or immune response (e.g.,neutralizing antibody response, T cell response (e.g., cytokineresponse)).

In some embodiments, an effective amount of a hMPV/hPIV3 RNA (e.g.,mRNA) vaccine is a dose that is reduced compared to the standard of caredose of a recombinant hMPV/hPIV3 protein vaccine. A “standard of care,”as provided herein, refers to a medical or psychological treatmentguideline and can be general or specific. “Standard of care” specifiesappropriate treatment based on scientific evidence and collaborationbetween medical professionals involved in the treatment of a givencondition. It is the diagnostic and treatment process that a physician/clinician should follow for a certain type of patient, illness orclinical circumstance. A “standard of care dose,” as provided herein,refers to the dose of a recombinant or purified hMPV/hPIV3 proteinvaccine, or a live attenuated or inactivated hMPV/hPIV3 vaccine, or ahMPV/hPIV3 VLP vaccine, that a physician/clinician or other medicalprofessional would administer to a subject to treat or preventhMPV/hPIV3, or a hMPV/hPIV3-related condition, while following thestandard of care guideline for treating or preventing hMPV/hPIV3, or ahMPV/hPIV3-related condition.

In some embodiments, the anti-hMPV/hPIV3 antigen antibody titer producedin a subject administered an effective amount of a hMPV/hPIV3 RNAvaccine is equivalent to an anti-hMPV/hPIV3 antigen antibody titerproduced in a control subject administered a standard of care dose of arecombinant or purified hMPV/hPIV3 protein vaccine, or a live attenuatedor inactivated hMPV/hPIV3 vaccine, or a hMPV/hPIV3 VLP vaccine.

In some embodiments, an effective amount of a hMPV/hPIV3 RNA (e.g.,mRNA) vaccine is a dose equivalent to an at least 2-fold reduction in astandard of care dose of a recombinant or purified hMPV/hPIV3 proteinvaccine. For example, an effective amount of a hMPV/hPIV3 RNA vaccinemay be a dose equivalent to an at least 3-fold, at least 4-fold, atleast 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, atleast 9-fold, or at least 10-fold reduction in a standard of care doseof a recombinant or purified hMPV/hPIV3 protein vaccine. In someembodiments, an effective amount of a hMPV/hPIV3 RNA vaccine is a doseequivalent to an at least at least 100-fold, at least 500-fold, or atleast 1000-fold reduction in a standard of care dose of a recombinant orpurified hMPV/hPIV3 protein vaccine. In some embodiments, an effectiveamount of a hMPV/hPIV3 RNA vaccine is a dose equivalent to a 2-, 3-, 4-,5-, 6-, 7-, 8-, 9-, 10-, 20-, 50-, 100-, 250-, 500-, or 1000-foldreduction in a standard of care dose of a recombinant or purifiedhMPV/hPIV3 protein vaccine. In some embodiments, the anti-hMPV/hPIV3antigen antibody titer produced in a subject administered an effectiveamount of a hMPV/hPIV3 RNA vaccine is equivalent to an anti-hMPV/hPIV3antigen antibody titer produced in a control subject administered thestandard of care dose of a recombinant or protein hMPV/hPIV3 proteinvaccine, or a live attenuated or inactivated hMPV/hPIV3 vaccine, or ahMPV/hPIV3 VLP vaccine. In some embodiments, an effective amount of ahMPV/hPIV3 RNA (e.g., mRNA) vaccine is a dose equivalent to a 2-fold to1000-fold (e.g., 2-fold to 100-fold, 10-fold to 1000-fold) reduction inthe standard of care dose of a recombinant or purified hMPV/hPIV3protein vaccine, wherein the anti-hMPV/hPIV3 antigen antibody titerproduced in the subject is equivalent to an anti-hMPV/hPIV3 antigenantibody titer produced in a control subject administered the standardof care dose of a recombinant or purified hMPV/hPIV3 protein vaccine, ora live attenuated or inactivated hMPV/hPIV3 vaccine, or a hMPV/hPIV3 VLPvaccine.

In some embodiments, the effective amount of a hMPV/hPIV3 RNA (e.g.,mRNA) vaccine is a total dose of 50-1000 μg. In some embodiments, theeffective amount of a hMPV/hPIV3 RNA (e.g., mRNA) vaccine is a totaldose of 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300,50-200, 50-100, 50-90, 50-80, 50-70, 50-60, 60-1000, 60-900, 60-800,60-700, 60-600, 60-500, 60-400, 60-300, 60-200, 60-100, 60-90, 60-80,60-70, 70-1000, 70-900, 70-800, 70-700, 70-600, 70-500, 70-400, 70-300,70-200, 70-100, 70-90, 70-80, 80-1000, 80-900, 80-800, 80-700, 80-600,80-500, 80-400, 80-300, 80-200, 80-100, 80-90, 90-1000, 90-900, 90-800,90-700, 90-600, 90-500, 90-400, 90-300, 90-200, 90-100, 100-1000,100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200,200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300,300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400,400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1000,500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600-900, 600-700,700-1000, 700-900, 700-800, 800-1000, 800-900, or 900-1000 μg. In someembodiments, the effective amount of a hMPV/hPIV3 RNA (e.g., mRNA)vaccine is a total dose of 50, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 μg. In someembodiments, the effective amount is a dose of 25-500 administered tothe subject a total of two times. In some embodiments, the effectiveamount of a hMPV/hPIV3 RNA (e.g., mRNA) vaccine is a dose of 25-500,25-400, 25-300, 25-200, 25-100, 25-50, 50-500, 50-400, 50-300, 50-200,50-100, 100-500, 100-400, 100-300, 100-200, 150-500, 150-400, 150-300,150-200, 200-500, 200-400, 200-300, 250-500, 250-400, 250-300, 300-500,300-400, 350-500, 350-400, 400-500 or 450-500 μg administered to thesubject a total of two times. In some embodiments, the effective amountof a hMPV/hPIV3 RNA (e.g., mRNA) vaccine is a total dose of 25, 50, 100,150, 200, 250, 300, 350, 400, 450, or 500 μg administered to the subjecta total of two times.

Vaccine efficacy may be assessed using standard analyses (see, e.g.,Weinberg et al., J Infect Dis. 2010 Jun. 1; 201(11):1607-10). Forexample, vaccine efficacy may be measured by double-blind, randomized,clinical controlled trials. Vaccine efficacy may be expressed as aproportionate reduction in disease attack rate (AR) between theunvaccinated (ARU) and vaccinated (ARV) study cohorts and can becalculated from the relative risk (RR) of disease among the vaccinatedgroup with use of the following formulas:

Efficacy=(ARU−ARV)/ARU×100; and

Efficacy=(1−RR)×100.

Likewise, vaccine effectiveness may be assessed using standard analyses(see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1;201(11):1607-10). Vaccine effectiveness is an assessment of how avaccine (which may have already proven to have high vaccine efficacy)reduces disease in a population. This measure can assess the net balanceof benefits and adverse effects of a vaccination program, not just thevaccine itself, under natural field conditions rather than in acontrolled clinical trial. Vaccine effectiveness is proportional tovaccine efficacy (potency) but is also affected by how well targetgroups in the population are immunized, as well as by othernon-vaccine-related factors that influence the ‘real-world’ outcomes ofhospitalizations, ambulatory visits, or costs. For example, aretrospective case control analysis may be used, in which the rates ofvaccination among a set of infected cases and appropriate controls arecompared. Vaccine effectiveness may be expressed as a rate difference,with use of the odds ratio (OR) for developing infection despitevaccination:

Effectiveness=(1−OR)×100.

In some embodiments, efficacy of the hMPV/hPIV3 vaccine is at least 60%relative to unvaccinated control subjects. For example, efficacy of thehMPV/hPIV3 vaccine may be at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 95%, at least 98%, or 100% relative tounvaccinated control subjects.

Sterilizing Immunity. Sterilizing immunity refers to a unique immunestatus that prevents effective pathogen infection into the host. In someembodiments, the effective amount of a hMPV/hPIV3 vaccine of the presentdisclosure is sufficient to provide sterilizing immunity in the subjectfor at least 1 year. For example, the effective amount of a hMPV/hPIV3vaccine of the present disclosure is sufficient to provide sterilizingimmunity in the subject for at least 2 years, at least 3 years, at least4 years, or at least 5 years. In some embodiments, the effective amountof a hMPV/hPIV3 vaccine of the present disclosure is sufficient toprovide sterilizing immunity in the subject at an at least 5-fold lowerdose relative to control. For example, the effective amount may besufficient to provide sterilizing immunity in the subject at an at least10-fold lower, 15-fold, or 20-fold lower dose relative to a control.

Detectable Antigen. In some embodiments, the effective amount of ahMPV/hPIV3 vaccine of the present disclosure is sufficient to producedetectable levels of hMPV/hPIV3 antigen as measured in serum of thesubject at 1-72 hours post administration. In some embodiments, theeffective amount of a hMPV/hPIV3 vaccine of the present disclosure issufficient to produce detectable levels of hMPV/hPIV3 antigen asmeasured in serum of the subject within 14 days (e.g., at 7-14 days)post administration.

Titer. An antibody titer is a measurement of the amount of antibodieswithin a subject, for example, antibodies that are specific to aparticular antigen (e.g., an anti-hMPV/hPIV3 antigen). Antibody titer istypically expressed as the inverse of the greatest dilution thatprovides a positive result. Enzyme-linked immunosorbent assay (ELISA) isa common assay for determining antibody titers, for example.

In some embodiments, the effective amount of a hMPV/hPIV3 vaccine of thepresent disclosure is sufficient to produce a 1,000-10,000 neutralizingantibody titer produced by neutralizing antibody against the hMPV/hPIV3antigen as measured in serum of the subject at 1-72 hours postadministration. In some embodiments, the effective amount is sufficientto produce a 1,000-5,000 neutralizing antibody titer produced byneutralizing antibody against the hMPV/hPIV3 antigen as measured inserum of the subject at 1-72 hours post administration. In someembodiments, the effective amount is sufficient to produce a5,000-10,000 neutralizing antibody titer produced by neutralizingantibody against the hMPV/hPIV3 antigen as measured in serum of thesubject at 1-72 hours post administration.

In some embodiments, the effective amount of a hMPV/hPIV3 vaccine of thepresent disclosure is sufficient to produce a 1,000-10,000 neutralizingantibody titer produced by neutralizing antibody against the hMPV/hPIV3antigen as measured in serum of the subject within 14 days of vaccineadministration. In some embodiments, the effective amount is sufficientto produce a 1,000-5,000 neutralizing antibody titer produced byneutralizing antibody against the hMPV/hPIV3 antigen as measured inserum of the subject within 14 days of vaccine administration. In someembodiments, the effective amount is sufficient to produce a5,000-10,000 neutralizing antibody titer produced by neutralizingantibody against the hMPV/hPIV3 antigen as measured in serum of thesubject within 14 days of vaccine administration.

In some embodiments, the neutralizing antibody titer is at least 100NT₅₀. For example, the neutralizing antibody titer may be at least 200,300, 400, 500, 600, 700, 800, 900 or 1000 NT₅₀. In some embodiments, theneutralizing antibody titer is at least 10,000 NT₅₀.

In some embodiments, the neutralizing antibody titer is at least 100neutralizing units per milliliter (NU/mL). For example, the neutralizingantibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or1000 NU/mL. In some embodiments, the neutralizing antibody titer is atleast 10,000 NU/mL.

In some embodiments, an anti-hMPV/hPIV3 antigen antibody titer producedin the subject is increased by at least 1 log relative to a control. Forexample, an anti-hMPV/hPIV3 antigen antibody titer produced in thesubject may be increased by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 logrelative to a control.

In some embodiments, an anti-hMPV/hPIV3 antigen antibody titer producedin the subject is increased at least 2 times relative to a control. Forexample, an anti-hMPV/hPIV3 antigen antibody titer produced in thesubject is increased by at least 3, 4, 5, 6, 7, 8, 9 or 10 timesrelative to a control.

In some embodiments, a geometric mean, which is the nth root of theproduct of n numbers, is generally used to describe proportional growth.Geometric mean, in some embodiments, is used to characterize antibodytiter produced in a subject.

A control may be, for example, an unvaccinated subject, or a subjectadministered a live attenuated hMPV/hPIV3 vaccine, an inactivatedhMPV/hPIV3 vaccine, or a protein subunit hMPV/hPIV3 vaccine.

EXAMPLES

The data provided below demonstrates that RNA vaccines comprisinghMPV/hPIV3 RNA polynucleotides, formulated in lipid nanoparticlescomprising Compound 25 of Formula (I), protect animals from challenge byboth viruses. Virus was not detectable in the lung or nose, and therewas no evidence of interference between vaccine components. Cotton ratswere completely protected from hPIV3 or hMPV after receiving 2 doses ofthe hMPV/hPIV3 RNA vaccine. hPIV3 F alone had higher neutralizationtiters than hPIV3 HN or the combination of hPIV3 HN and hPIV3 F.Further, seronegative monkeys were completely protected from hMPV andhPIV3 challenge after 2 doses of the hMPV/hPIV3 RNA vaccine. Innaturally seropositive animals, a single dose of vaccine boosted hMPVand hPIV3 titers 4-10 fold. Again, hPIV3 F alone had higherneutralization titers than hPIV3 HN or the combination of hPIV3 HN andhPIV3 F.

Example 1 Manufacture of Polynucleotides

According to the present disclosure, the manufacture of polynucleotidesand/or parts or regions thereof may be accomplished utilizing themethods taught in International Publication WO2014/152027, entitled“Manufacturing Methods for Production of RNA Transcripts,” the contentsof which is incorporated herein by reference in its entirety.

Purification methods may include those taught in InternationalPublication WO2014/152030 and International Publication WO2014/152031,each of which is incorporated herein by reference in its entirety.

Detection and characterization methods of the polynucleotides may beperformed as taught in International Publication WO2014/144039, which isincorporated herein by reference in its entirety.

Characterization of the polynucleotides of the disclosure may beaccomplished using polynucleotide mapping, reverse transcriptasesequencing, charge distribution analysis, detection of RNA impurities,or any combination of two or more of the foregoing. “Characterizing”comprises determining the RNA transcript sequence, determining thepurity of the RNA transcript, or determining the charge heterogeneity ofthe RNA transcript, for example. Such methods are taught in, forexample, International Publication WO2014/144711 and InternationalPublication WO2014/144767, the content of each of which is incorporatedherein by reference in its entirety.

Example 2 Chimeric Polynucleotide Synthesis

According to the present disclosure, two regions or parts of a chimericpolynucleotide may be joined or ligated using triphosphate chemistry. Afirst region or part of 100 nucleotides or less is chemicallysynthesized with a 5′ monophosphate and terminal 3′ desOH or blocked OH,for example. If the region is longer than 80 nucleotides, it may besynthesized as two strands for ligation.

If the first region or part is synthesized as a non-positionallymodified region or part using in vitro transcription (IVT), conversionthe 5′monophosphate with subsequent capping of the 3′ terminus mayfollow.

Monophosphate protecting groups may be selected from any of those knownin the art.

The second region or part of the chimeric polynucleotide may besynthesized using either chemical synthesis or IVT methods. IVT methodsmay include an RNA polymerase that can utilize a primer with a modifiedcap. Alternatively, a cap of up to 130 nucleotides may be chemicallysynthesized and coupled to the IVT region or part.

For ligation methods, ligation with DNA T4 ligase, followed by treatmentwith DNase should readily avoid concatenation.

The entire chimeric polynucleotide need not be manufactured with aphosphate-sugar backbone. If one of the regions or parts encodes apolypeptide, then such region or part may comprise a phosphate-sugarbackbone.

Ligation is then performed using any known click chemistry, orthoclickchemistry, solulink, or other bioconjugate chemistries known to those inthe art.

Synthetic Route

The chimeric polynucleotide may be made using a series of startingsegments. Such segments include:

(a) a capped and protected 5′ segment comprising a normal 3′OH (SEG. 1)

(b) a 5′ triphosphate segment, which may include the coding region of apolypeptide and a normal 3′OH (SEG. 2)

(c) a 5′ monophosphate segment for the 3′ end of the chimericpolynucleotide (e.g., the tail) comprising cordycepin or no 3′OH (SEG.3)

After synthesis (chemical or IVT), segment 3 (SEG. 3) may be treatedwith cordycepin and then with pyrophosphatase to create the 5′monophosphate.

Segment 2 (SEG. 2) may then be ligated to SEG. 3 using RNA ligase. Theligated polynucleotide is then purified and treated with pyrophosphataseto cleave the diphosphate. The treated SEG.2-SEG. 3 construct may thenbe purified and SEG. 1 is ligated to the 5′ terminus. A furtherpurification step of the chimeric polynucleotide may be performed.

Where the chimeric polynucleotide encodes a polypeptide, the ligated orjoined segments may be represented as: 5′UTR (SEG. 1), open readingframe or ORF (SEG. 2) and 3′UTR+PolyA (SEG. 3).

The yields of each step may be as much as 90-95%.

Example 3 PCR for cDNA Production

PCR procedures for the preparation of cDNA may be performed using 2×KAPA HIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). Thissystem includes 2× KAPA ReadyMix 12.5 μl; Forward Primer (10 μM) 0.75μl; Reverse Primer (10 μM) 0.75 μl; Template cDNA 100 ng; and dH₂0diluted to 25.0 μl. The reaction conditions may be at 95° C. for 5 min.The reaction may be performed for 25 cycles of 98° C. for 20 sec, then58° C. for 15 sec, then 72° C. for 45 sec, then 72° C. for 5 min, then4° C. to termination.

The reaction may be cleaned up using Invitrogen's PURELINK™ PCR MicroKit (Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg).Larger reactions may require a cleanup using a product with a largercapacity. Following the cleanup, the cDNA may be quantified using theNANODROP™ and analyzed by agarose gel electrophoresis to confirm thatthe cDNA is the expected size. The cDNA may then be submitted forsequencing analysis before proceeding to the in vitro transcriptionreaction.

Example 4 In Vitro Transcription (IVT)

The in vitro transcription reaction generates RNA polynucleotides. Suchpolynucleotides may comprise a region or part of the polynucleotides ofthe disclosure, including chemically modified RNA (e.g., mRNA)polynucleotides. The chemically modified RNA polynucleotides can beuniformly modified polynucleotides. The in vitro transcription reactionutilizes a custom mix of nucleotide triphosphates (NTPs). The NTPs maycomprise chemically modified NTPs, or a mix of natural and chemicallymodified NTPs, or natural NTPs.

A typical in vitro transcription reaction includes the following:

1) Template cDNA 1.0 μg 2) 10x transcription buffer 2.0 μl (400 mMTris-HCl pH 8.0, 190 mM MgCl₂, 50 mM DTT, 10 mM Spermidine) 3) CustomNTPs (25 mM each) 0.2 μl 4) RNase Inhibitor 20 U 5) T7 RNA polymerase3000 U 6) dH₂0 up to 20.0 μl. and 7) Incubation at 37° C. for 3 hr-5hrs.

The crude IVT mix may be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase may then be used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA may bepurified using Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA polynucleotide may be quantified usingthe NanoDrop and analyzed by agarose gel electrophoresis to confirm theRNA polynucleotide is the proper size and that no degradation of the RNAhas occurred.

Example 5 Enzymatic Capping

Capping of a RNA polynucleotide is performed as follows where themixture includes: IVT RNA 60 μg-180 μg and dH₂O up to 72 μl. The mixtureis incubated at 65° C. for 5 minutes to denature RNA, and then istransferred immediately to ice.

The protocol then involves the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂0 (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The RNA polynucleotide may then be purified using Ambion's MEGACLEAR™Kit (Austin, Tex.) following the manufacturer's instructions. Followingthe cleanup, the RNA may be quantified using the NANODROP™(ThermoFisher, Waltham, Mass.) and analyzed by agarose gelelectrophoresis to confirm the RNA polynucleotide is the proper size andthat no degradation of the RNA has occurred. The RNA polynucleotideproduct may also be sequenced by running a reverse-transcription-PCR togenerate the cDNA for sequencing.

Example 6 PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This is done by mixingcapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂) (12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂O up to 123.5 μl and incubation at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction may be skipped and proceed directly to cleanup withAmbion's MEGACLEAR™ kit (Austin, Tex.) (up to 500 μg). Poly-A Polymerasemay be a recombinant enzyme expressed in yeast.

It should be understood that the processivity or integrity of the polyAtailing reaction may not always result in an exact size polyA tail.Hence, polyA tails of approximately between 40-200 nucleotides, e.g.,about 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 150-165, 155, 156,157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scope ofthe present disclosure.

Example 7 Natural 5′ Caps and 5′ Cap Analogues

5′-capping of polynucleotides may be completed concomitantly during thein vitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3″-O-Me-m7G(5)ppp(5′) G [the ARCAcap];G(5)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of modified RNA may becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). Cap 1 structure may be generated using both VacciniaVirus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure may be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes are preferably derived from a recombinantsource.

When transfected into mammalian cells, the modified mRNAs have astability of between 12-18 hours or more than 18 hours, e.g., 24, 36,48, 60, 72 or greater than 72 hours.

Example 8 Capping Assays Protein Expression Assay

Polynucleotides (e.g., mRNA) encoding a polypeptide, containing any ofthe caps taught herein, can be transfected into cells at equalconcentrations. The amount of protein secreted into the culture mediumcan be assayed by ELISA at 6, 12, 24 and/or 36 hours post-transfection.Synthetic polynucleotides that secrete higher levels of protein into themedium correspond to a synthetic polynucleotide with a highertranslationally-competent cap structure.

Purity Analysis Synthesis

RNA (e.g., mRNA) polynucleotides encoding a polypeptide, containing anyof the caps taught herein can be compared for purity using denaturingAgarose-Urea gel electrophoresis or HPLC analysis. RNA polynucleotideswith a single, consolidated band by electrophoresis correspond to thehigher purity product compared to polynucleotides with multiple bands orstreaking bands. Chemically modified RNA polynucleotides with a singleHPLC peak also correspond to a higher purity product. The cappingreaction with a higher efficiency provides a more pure polynucleotidepopulation.

Cytokine Analysis

RNA (e.g., mRNA) polynucleotides encoding a polypeptide, containing anyof the caps taught herein can be transfected into cells at multipleconcentrations. The amount of pro-inflammatory cytokines, such asTNF-alpha and IFN-beta, secreted into the culture medium can be assayedby ELISA at 6, 12, 24 and/or 36 hours post-transfection. RNApolynucleotides resulting in the secretion of higher levels ofpro-inflammatory cytokines into the medium correspond to apolynucleotides containing an immune-activating cap structure.

Capping Reaction Efficiency

RNA (e.g., mRNA) polynucleotides encoding a polypeptide, containing anyof the caps taught herein can be analyzed for capping reactionefficiency by LC-MS after nuclease treatment. Nuclease treatment ofcapped polynucleotides yield a mixture of free nucleotides and thecapped 5′-5-triphosphate cap structure detectable by LC-MS. The amountof capped product on the LC-MS spectra can be expressed as a percent oftotal polynucleotide from the reaction and correspond to cappingreaction efficiency. The cap structure with a higher capping reactionefficiency has a higher amount of capped product by LC-MS.

Example 9 Agarose Gel Electrophoresis of Modified RNA or RT PCR Products

Individual RNA polynucleotides (200-400 ng in a 20 μl volume) or reversetranscribed PCR products (200-400 ng) may be loaded into a well on anon-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, Calif.) and runfor 12-15 minutes, according to the manufacturer protocol.

Example 10 Nanodrop Modified RNA Quantification and UV Spectral Data

Chemically modified RNA polynucleotides in TE buffer (1 μl) are used forNanodrop UV absorbance readings to quantitate the yield of eachpolynucleotide from an chemical synthesis or in vitro transcriptionreaction.

Example 11 Formulation of Modified mRNA Using Lipidoids

RNA (e.g., mRNA) polynucleotides may be formulated for in vitroexperiments by mixing the polynucleotides with the lipidoid at a setratio prior to addition to cells. In vivo formulation may require theaddition of extra ingredients to facilitate circulation throughout thebody. To test the ability of these lipidoids to form particles suitablefor in vivo work, a standard formulation process used for siRNA-lipidoidformulations may be used as a starting point. After formation of theparticle, polynucleotide is added and allowed to integrate with thecomplex. The encapsulation efficiency is determined using a standard dyeexclusion assays.

Example 12 Expression of hMPV and hPIV3 Fusion Protein on Cell Surface

The instant study was designed to show that hMPV/hPIV3 mRNA vaccinesencoding the hMPV F protein and the hPIV3 F protein led to cell surfaceexpression of the antigen in cultured Hela cells. The mRNA constructswere transfected into Hela cells. Expression of the hMPV F protein wasdetected by fluorescent staining using hMPV-F-specific antibodies MPE-8(Table 1). Untransfected cells (mock) were also stained as negativecontrol. In untransfected cells or cells stained only with secondaryantibodies, no hMPV F protein signal was detected, while hMPV F proteinsignal was detected in transfected cells stained with MPE-8 antibodies(FIG. 1A). Expression of the hPIV3 F protein was detected by stainingusing the hPIV3-F-specific antibodies MAB10207 and surface expression ofhPIV3 protein was detected (FIG. 1B)

TABLE 1 Fluorescent Staining of Cells Transfected with hMPV/HPIV3 mRNAVaccine Constructs Sample Mean FL4-H Mock-unstained 494.38Mock-Secondary only 3,944.18 Mock-MPE8 (500 nM) 5,452.55 hMPV-Secondaryonly 2,160.82 hMPV-MPE8 (500 nM) 617,307.76

Example 13 hMPV/hPIV3 Cotton Rat Challenge

The instant study was designed to show that the hMPV/hPIV3 mRNA vaccineconstructs encoding the hMPV F protein and the hPIV3 F protein inducedhigh levels of neutralizing antibodies in cotton rats and reduced theviral titers in the nose and lungs of the immunized cotton rats afterchallenge with hMPV or hPIV3 viruses. The study design is shown in Table2. Animals were dosed on Days 0 and 28 and were challenged on Day 56.Animals were bled on Days −1, 27 and 56. Viral titers were determined 5days post challenge.

Cotton rats that are negative for hMPV and hPIV3 were divided into 19groups (n=8), and each group was vaccinated with 2 doses of mRNAvaccines on days 0 and 28. The mRNA vaccines were formulated in eitherMC3 lipids or Compound 1 lipids. Immunized cotton rats were challengedwith a lethal dose of hMPV or hPIV3 on day 57 post immunization. Theviral titers in the nose and lungs of the challenged cotton rats weremeasured on day 5 post challenge and the serum neutralizing antibodytiters were measured one day prior to immunization, and on days 27 and56 post immunization.

All cotton rats receiving 2 doses of hMPV/HPIV3 mRNA vaccine werecompletely protected from hMPV or hPIV3 infection, and the twoformulations with either MC3 or Compound 1 lipids yielded similar levelsof protection (FIGS. 2A-2B). Neutralizing antibody titers in the sera ofthe immunized cotton rats were analyzed. The results show that thehMPV/HPIV3 mRNA vaccines induced high levels of neutralizing antibodiesagainst hMPV (FIG. 3A) or hPIV3 (FIG. 3B) in immunized cotton rat.Further, mRNA vaccine formulations with Compound 1 or MC3 lipids inducedcomparable levels of neutralizing antibodies against hMPV (FIG. 3A,compare Group 2 with Group 7). A control group immunized with Fl-hMPVshowed very low neutralizing antibody titers as expected (FIG. 3A, Group6). Similarly, mRNA vaccine formulations with Compound 1 or MC3 lipidsinduced comparable levels of neutralizing antibodies against hPIV3 (FIG.3, compare Group 9 with Group 16). MRNA vaccines encoding hPIV3 Fprotein induced neutralizing antibody titers that is 3-fold higher thanthat of mRNA vaccines encoding hPIV3 HN protein at the same dosage (25μg) (FIG. 3B, compare Group 10 with Group 11). Further, the presence ofmRNA constructs in the mRNA vaccine encoding hMPV antigen does notinterfere with the immunogenicity of mRNA constructs encoding hPIV3antigen (FIG. 3B, compare Group 9 with Group 12).

TABLE 2 Cotton Rat Challenge Study Design Cotton Dose Challenge RatGroup n Vaccine (μg) Formulation Vaccine (Day 57) Endpoint 1 8hMPV/PIV/RSV 10/10/30 Compound 1 D 0, D 28 hMPV Lung and Nose 2 8hMPV/PIV 25/25 Compound 1 D 0, D 28 viral titer: Day 3 8 hMPV 25Compound 1 D 0, D 28 5 post challenge 4 8 hMPV 10 Compound 1 D 0, D 28and Neutralizing 5 8 PBS NA NA D 0, D 28 antibody titer 6 8 FI-hMPV NANA D 0, D 28 (Day-1, D 27, D 56) 7 8 hMPV/PIV 25/25 MC3 D 0, D 28 8 8hMPV/PIV/RSV 10/10/30 Compound 1 D 0, D 28 PIV3 9 8 hMPV/PIV 25/25Compound 1 D 0, D 28 10 8 hMPV/PIV-F 25/25 Compound 1 D 0, D 28 11 8hMPV/PIV-HN 25/25 Compound 1 D 0, D 28 12 8 PIV 25 Compound 1 D 0, D 2813 8 PIV 10 Compound 1 D 0, D 28 14 8 PBS NA NA D 0, D 28 15 8 FI-PIV3NA NA D 0, D 28 16 8 hMPV/PIV 25/25 MC3 D 0, D 28 17 8 hMPV/PIV/RSV10/10/30 Compound 1 D 0, D 28 RSV 18 8 RSV 30 Compound 1 D 0, D 28 19 8PBS NA NA D 0, D 28

Example 14 Safety and Efficacy of hMPV/hPIV3 Vaccination in Cotton RatChallenge

The instant study was designed to evaluate the safety and efficacy ofhuman metapneumovirus (hMPV)/parainfluenza 3 (PIV3) mRNA vaccines in thecotton rat model of hMPV or PIV3 challenge. Lipid nanoparticle(LNP)-formulated combinations of mRNA encoding the following antigens:

-   -   hMPV fusion (F) protein (Strain: A/TN92-4) (SEQ ID NO: 4)    -   PIV3 F protein (strain: PER/FLA4815/2008) (SEQ ID NO: 5)    -   PIV3 hemagglutinin-neuraminidase (HN) protein        (Strain: 612507167) (SEQ ID NO: 6)

The mRNAs were formulated in a mixture of 4 lipids, including Compound1; 1,2-dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG2000-DMG);1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and cholesterol.

The combination vaccine includes hMPV-F and PIV3-F mRNA co-formulated ata 1:1 mass ratio in LNP.

Groups of 8 female cotton rats were immunized with different dose levelsof LNP-formulated mRNAs encoding hMPV-F, PIV3-F, and PIV3-HN, eitheralone or in combination, as indicated in Table 3. Control groups wereinoculated with phosphate-buffered saline (PBS), formalin-inactivated(FI)-hMPV, or FI-PIV3. FI-hMPV and FI-PIV3 were produced, and are usedas positive controls for vaccine-mediated enhanced respiratory disease(ERD) after hMPV or PIV3 challenge, respectively. All animals wereimmunized intramuscularly (IM) on Days 0 and 28. Serum was collected onDay 56 for measurement of neutralizing antibody titers to hMPV (Groups2-6) or PIV3 (Groups 9-15) by 60% plaque reduction neutralization test(PRNT). Animals were challenged intra-nasally on Day 56 with either 10⁵pfu hMPV/A2 (Groups 2-6) or 10⁵ pfu PIV3 (Groups 9-15). Five days later(Day 61) nasal tissue and lungs were collected. Viral load in bothtissues was determined by plaque assay and pulmonary histopathology wasevaluated on hematoxylin and eosin (H&E) stained fixed lung sections.Parameters of pulmonary inflation include interstitial pneumonia(inflammatory cell infiltration and thickening of alveolar walls) andalveolitis (cells within the alveolar spaces). Sections were evaluatedon a 0-4 severity scale and subsequently converted to a 0-100%histopathology scale.

TABLE 3 mRNAdose (μg) Vaccine Challenge Group n Vaccine total hMPV-FPIV3-F PIV3-HN Schedule (Day 56) 2 8 hMPV-F/PIV3-F/PIV3-HN 50 25 12.512.5 Day 0 & 28 hMPV 3 8 hMPV-F 25 25 0 0 Day 0 & 28 hMPV 4 8 hMPV-F 1010 0 0 Day 0 & 28 hMPV 5 8 PBS NA NA NA NA Day 0 & 28 hMPV 6 8 FI-hMPVNA NA NA NA Day 0 & 28 hMPV 9 8 hMPV-F/PIV3-F/PIV3-HN 50 25 12.5 12.5Day 0 & 28 PIV3 10 8 hMPV-F/PIV3-F 50 25 25 0 Day 0 & 28 PIV3 11 8hMPV-F/PIV3-HN 50 25 0 25 Day 0 & 28 PIV3 12 8 PIV3-F/PIV3-HN 25  0 12.512.5 Day 0 & 28 PIV3 13 8 PIV3-F/PIV3-HN 10  0 5 5 Day 0 & 28 PIV3 14 8PBS NA NA NA NA Day 0 & 28 PIV3 15 8 FI-PIV3 NA NA NA NA Day 0 & 28 PIV3NA—not applicablehMPV Lung and Nose Viral Titration

Lung and nose homogenates were clarified by centrifugation and dilutedin EMEM. Confluent MK-2 monolayers were infected in duplicates withdiluted homogenates in 24 well plates. After one hour incubation at 37°C. in a 5% CO₂ incubator, the wells were overlaid with 0.75%methylcellulose medium. After 7 days of incubation, the overlays wereremoved and the cells were fixed for one hour and air dried forimmune-staining. Upon blocking the wells, mouse anti-hMPV nucleoprotein(N) at a 1:1000 dilution to each well, followed by horseradishperoxidase (HRP) conjugated goat anti-mouse IgG diluted at 1:1000.TrueBlue peroxidase substrate was added to each well and incubated atroom temperature for 10 to 15 minutes. Visible plaques were counted andvirus titers were expressed as plaque forming units (pfu) per gram (g)of tissue. Viral titers were reported as mean+standard deviation (SD) oflog 10 transformed values for all animals in a group.

PIV3 Lung and Nose Viral Titration

Lung and nose homogenates were clarified by centrifugation and dilutedin EMEM. Confluent MA-104 (monkey kidney cells) monolayers were infectedin duplicates with diluted homogenates in 24 well plates. After two-hourincubation at 37° C. in a 5% CO₂ incubator, the wells were overlaid with0.75% methylcellulose medium. After 4 days of incubation, the overlayswere removed and the cells were fixed and stained with 0.1% crystalviolet for one hour and then rinsed and air dried. Plaques were countedand virus titers were expressed as pfu/g of tissue. Viral loads werereported as mean+SD of log 10 transformed values for all animals in agroup.

hMPV Neutralizing Antibody Assay

Heat inactivated sera samples were diluted 1:10 with EMEM and seriallydiluted further 1:4. Diluted serum samples were incubated with hMPV/A2(25-50 pfu) for 1 hour at room temperature and inoculated in duplicatesonto confluent MK-2 monolayers in 24 well plates. After one hourincubation at 37° C. in a 5% CO₂ incubator, the wells were overlaid with0.75% Methylcellulose medium. After 7 days of incubation, the overlayswere removed and the cells were fixed in cold acetone/methanol solutionfor one hour and air dried for immune-staining. Upon blocking the wellswith blotto, mouse anti-hMPV N protein at a 1:1000 dilution to eachwell, followed by HRP conjugated goat anti-mouse IgG diluted at 1:1000.TrueBlue peroxidase substrate was added to each well and incubated atroom temperature for 10 to 15 minutes. Visible plaques were counted andthe corresponding reciprocal neutralizing antibody titers weredetermined at the 60% reduction end-point of the virus control using thestatistics program “plqrd.manual.entry”. Neutralizing titers werereported as mean+/−SD of log 2 transformed titers for all animals in agroup.

PIV3 Neutralizing Antibody Assay (60% Reduction)

Heat inactivated sera samples were diluted 1:10 with EMEM and seriallydiluted further 1:4. Diluted serum samples were incubated with PIV3(25-50 pfu) for 1 hour at room temperature and inoculated in duplicatesonto confluent MA-104 monolayers in 24 well plates. After two-hourincubation at 37° C. in a 5% CO₂ incubator, the wells were overlaid with0.75% Methylcellulose medium. After 4 days of incubation, the overlayswere removed and the cells were fixed and stained with 0.1% crystalviolet for one hour and then rinsed and air dried. The correspondingreciprocal neutralizing antibody titers were determined at the 60%reduction end-point of the virus control using a statistics program.Neutralizing titers were reported as mean+SD of log 2 transformed titersfor all animals in a group.

Pulmonary Histopathology

Lungs were dissected and inflated with 10% neutral buffered formalin totheir normal volume, and then immersed in the same fixative solution.Following fixation, the lungs were embedded in paraffin, sectioned andstained with H&E. Four parameters of pulmonary inflammation wereevaluated: peribronchiolitis (inflammatory cell infiltration around thebronchioles), perivasculitis (inflammatory cell infiltration around thesmall blood vessels), interstitial pneumonia (inflammatory cellinfiltration and thickening of alveolar walls), and alveolitis (cellswithin the alveolar spaces). Slides were scored blind on a 0-4 severityscale. The scores were subsequently converted to a 0-100% histopathologyscale, and reported as mean+SD of all animals in a group.

hMPV neutralizing antibody titers were measured in Day 56 serum fromGroups 2-6 (FIG. 4A) and were detected at high levels in all animalsimmunized with hMPV-F mRNA (Groups 2-4) as well as at low levels inanimals immunized with FI-hMPV (Group 6). There was a small doseresponse to the monovalent hMPV-F vaccine (Group 3 vs. Group 4).Neutralizing antibody titers were similar in Groups 2 and 3,demonstrating no interference of the anti-hMPV antibody response by mRNAencoding hPIV3 proteins.

PIV3 neutralizing antibody titers were measured in Day 56 serum fromGroups 9-15 (FIG. 4B) and were detected at high levels in all animalsimmunized with PIV3-F and/or PIV3-HN mRNA (Groups 9-13) as well as atlow levels in animals immunized with FI-PIV3 (Group 15). There was asmall dose response to the PIV3-F/PIV3-HN vaccine (Group 12 vs. Group13). Neutralizing antibody titers were similar in Groups 9 and 12,demonstrating no interference of the anti-PIV3 antibody response by mRNAencoding hMPV-F. The PIV3-F vaccine elicited higher antibody titers thanthe PIV3-HN vaccine (Group 10 vs. Group 11), suggesting that the PIV3neutralizing antibody responses induced by the vaccines containing bothPIV3-F and PIV3-HN (Groups 9, 12, and 13) were likely dominatedprimarily by the response to PIV3-F. Among the combination vaccinegroups tested, Group 10, the combination of PIV3-F/hMPV-F showed thehighest level of PIV3 Neutralizing antibody titers.

The ability of the hMPV/PIV3 mRNA vaccines to protect against challengewas determined by measuring viral load in lung and nose from immunizedanimals 5 days after hMPV intranasal challenge (Groups 2-6) (FIG. 5A) orPIV3 challenge (Groups 9-15) (FIG. 5B). High level of virus was detectedin PBS control animals (Groups 5 and 14), but was close to or below thelimit of quantification in all mRNA-immunized animals (Groups 2-4 and9-13), demonstrating full protection in both the lung and the nose. TheFI-hMPV vaccine (Group 6) afforded only a small level of protectionagainst hMPV challenge, and the FI-PIV3 vaccine (Group 15) provided noprotection against PIV3 challenge.

Lung pathology was scored in tissues obtained 5 days after hMPVintranasal challenge (Groups 2-6) (FIG. 6A) or PIV3 challenge (Groups9-15) (FIG. 6B). All mRNA-immunized animals (Groups 2-4 and 9-13)exhibited lung histopathology scores equivalent to the PBS controls(Groups 5 and 14), indicating no vaccine-enhanced respiratory disease(ERD). In contrast, animals vaccinated with the FI-hMPV (Group 6) orFI-PIV3 (Group 15) positive controls demonstrated elevated levels ofboth alveolitis and interstitial pneumonia after hMPV or PIV3 challenge,respectively.

Example 15 hMPV/hPIV3 African Green Monkey Challenge

The instant study was designed to show that the hMPV/HPIV3 mRNA vaccineconstructs encoding the hMPV F protein and the hPIV3 F protein inducedhigh levels of neutralizing antibodies in African green monkeys andreduced the viral titers in the nose and lungs of the immunized Africangreen monkeys after challenge with hMPV or hPIV3 viruses. The studydesign is shown in Table 4 (G=Group).

Sero-negative (to both hMPV and hPIV3) African green monkeys weredivided into 10 groups (n=3) and hMPV sero-positive or hPIV3sero-positive African green monkeys were divided into 3 groups,respectively (n=3). Each sero-negative group was vaccinated with 2 dosesof mRNA vaccines on days 0 and 28. Each sero-positive group wasvaccinated with 1 dose of mRNA vaccines on day 0. The mRNA vaccines wereformulated in either MC3 lipids or Compound 1 lipids. All African greenmonkeys were monitored for injection site reactions 4 hours, 24 hours,and 72 hours after each injection and no erythema or edema was observedin any African green monkey.

Immunized sero-negative African green monkeys were challenged with alethal dose of hMPV or hPIV3 on day 57 post immunization. The viraltiters in the nose and lungs of the challenged sero-negative Africangreen monkeys were measured on day 5 post challenge and the serumneutralizing antibody titers were measured one day prior toimmunization, and on days 27 and 56 post immunization. Serumneutralizing antibody titers of sero-positive African green monkeys weremeasured 28 days prior to immunization, on the day of immunizations, anddays 14, 42, and 56 post immunization.

Sero-negative African green monkeys were completely protected from hMPVand hPIV3 infection after two doses of the hMPV/HPIV3 mRNA vaccine(FIGS. 7A-7B). Serum neutralizing antibody titers induced by thehMPV/HPIV3 mRNA vaccines in African green monkeys were analyzed. Theresults show that in sero-negative African green monkeys, two doses of200 μg or 100 μg mRNA vaccine induced high levels of neutralizingantibody titers against hMPV (FIGS. 8A and 8B, and FIG. 13A), while twodoses of 10μ mRNA vaccine induced lower levels of neutralizing antibodytiters against hMPV (FIG. 8C). In hMPV sero-positive African greenmonkeys, one dose of 200 μg or 50 μg mRNA vaccine induced high levels ofneutralizing antibody titers against hMPV (FIGS. 9B and 9C), while theplacebo did not induce neutralizing antibodies (FIG. 9A). Similarly, insero-negative African green monkeys, two doses of 200 μg, 100 μg, or 10μg mRNA vaccine induced high levels of neutralizing antibody titersagainst hPIV3 (FIGS. 10A-10C, and FIG. 11A). In hPIV3 sero-positiveAfrican green monkeys, one dose of 200 μg or 50 μg mRNA vaccine inducedhigh levels of neutralizing antibody titers against hPIV3 (FIGS. 12B and12C), while the placebo did not induce neutralizing antibodies (FIG.12A). Further, mRNA vaccines encoding hPIV3 F protein inducedneutralizing antibody titers that is 3-fold higher than that of mRNAvaccines encoding hPIV3 HN protein at the same dosage (FIGS. 11A-11B).However, at day 56 post immunization, neutralizing antibody titerinduced by hPIV3 F protein or HN protein became comparable (FIG. 14A).hPIV3 mRNA vaccine alone induced comparable neutralizing antibody titersas the hMPV/HPIV3 mRNA vaccine, indicating that hPIV3 F protein alone issufficient to generate strong protective response against hPIV3.

For sero-positive African green monkeys, a single dose of hMPV/HPIV3mRNA vaccine was able to boost neutralizing antibody titers against hMPVby 8-10 fold (FIG. 13B), and against hPIV3 by 4-10 fold (FIG. 14B) 42days post immunization.

TABLE 4 African Green Monkey Challenge Study Design Challenge G nVaccine Dose Formulation Vaccine (Day 57) Endpoint hMPV 1 3 hMPV/PIV100/100 Compound 1 D 0, D 28 hMPV Neutralizing Nose and sero- 2 3hMPV/PIV 50/50 Compound 1 D 0, D 28 and total Trachea positive 3 3hMPV/PIV 5/5 Compound 1 D 0, D 28 IgG titer: viral PIV3 4 3 PBS NA NA D0, D 28 Prebleeds, titer by sero- 5 3 hMPV/PIV 100/100 Compound 1 D 0, D28 PIV3 Day 27, RT-PCR positive 6 3 hMPV/PIV 50/50 Compound 1 D 0, D 28Day 56 7 3 hMPV/PIV 5/5 Compound 1 D 0, D 28 8 3 hMPV/PIV-F 50/50Compound 1 D 0, D 28 9 3 hMPV/PIV-HN 50/50 SM012 D 0, D 28 10 3 PBS NANA D 0, D 28 11 3 PBS NA NA D 0 Neutralizing antibody titers: 12 3hMPV/PIV 100/100 Compound 1 D 0 Day 28, 0, 14, 42, 56 13 3 hMPV/PIV25/25 Compound 1 D 0 14 3 PBS NA NA D 0 15 3 hMPV/PIV 100/100 Compound 1D 0 16 3 hMPV/PIV 25/25 Compound 1 D 0

Example 16 Immunogenicity of hMPV/hPIV3 mRNA Vaccines in African GreenMonkeys

Lipid nanoparticle (LNP)—formulated combination of mRNA encoding thefollowing antigens:

-   -   hMPV Fusion (F) protein(Strain: A/TN92-4) (SEQ ID NO: 4)    -   PIV3 F protein (strain: PER/FLA4815/2008) (SEQ ID NO: 5)    -   PIV3 hemagglutinin-neuraminidase (HN) protein        (Strain: 612507167) (SEQ ID NO: 6)

The mRNAs were formulated in a mixture of 4 lipids, including Compound1; 1,2-dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG2000-DMG);1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and cholesterol.

The immunogenicity of the LNP-formulated hMPV/PIV3-F/PIV3-HN mRNAvaccine was evaluated in African Green Monkeys that had beenexperimentally infected with hMPV or PIV3 previously, and therefore hadserum hMPV or PIV3 neutralizing antibody titers prior to vaccination.African green monkeys previously exposed to hMPV or PIV3 provide a modelof immune memory recall responses to vaccination that is intended tomimic the responses that can be anticipated in seropositive humanadults. Groups of three hMPV-exposed (Groups 11-13) or PIV3-exposed(Groups 14-16) AGM were immunized intramuscularly (IM) a single time onDay 0 with different dose levels of the hMPV-F/PIV3-F/PIV3-HN vaccine orwith a phosphate-buffered saline (PBS) control, as indicated in Table 5.The results in previous Example 15 show that PIV3-HN contributesminimally to the PIV3 neutralizing antibody response. Serum wascollected 28 days before immunization (Day −28), the day of immunization(Day 0), and 14, 42, and 56 days after immunization. Serum neutralizingantibody titers to hMPV (Groups 11-13) or PIV3 (Groups 14-16) weremeasured by 60% plaque reduction neutralization test (PRNT).

TABLE 5 previous mRNA dose (μg) Vaccine Group n infection Vaccine totalhMPV-F PIV-F PIV3-HN Schedule 11 3 hMPV PBS NA NA NA NA Day 0 12 3 hMPVhMPV-F/PIV3-F/PIV3-HN 200  100  50 50 Day 0 13 3 hMPVhMPV-F/PIV3-F/PIV3-HN 50 25 12.5 12.5 Day 0 14 3 PIV3 PBS NA NA NA NADay 0 15 3 PIV3 hMPV-F/PIV3-F/PIV3-HN 200  100  50 50 Day 0 16 3 PIV3hMPV-F/PIV3-F/PIV3-HN 50 25 12.5 12.5 Day 0 NA—not applicablehMPV Neutralizing Antibody Assay

Heat inactivated sera samples are diluted 1:10 and serially dilutedfurther 1:4. Diluted serum samples are incubated with hMPV/A2 (25-50plaque forming units (pfu)) for 1 hour at room temperature andinoculated in duplicates onto confluent MK-2 monolayers in 24-wellplates. After one hour incubation at 37° C. in a 5% CO₂ incubator, thewells are overlaid with 0.75% Methylcellulose medium. After 7 days ofincubation, the overlays are removed and washed once in PBS. The cellsare fixed in cold acetone/methanol solution for one hour and air driedfor immuno-staining. The cells are permeabilized in 0.4% Triton-Xsolution and incubated in blocking solution (10% bovine serum albumin).Mouse anti-hMPV nucleoprotein (N) at a 1:1,000 dilution is added to eachwell, followed by horseradish peroxidase (HRP) conjugated rabbitanti-mouse IgG diluted at 1:5,000. AEC chromogen detection solution isused for coloration after two hours of incubation or until red plaquesare visible. Plaques are counted and virus titers are expressed asplaque forming units. The corresponding reciprocal neutralizing antibodytiters are determined at the 60% reduction end-point of the viruscontrol using a statistics program. Neutralizing titers are reported asmean+/−SD of log2 transformed titers for all animals in a group at agiven time point.

PIV3 Neutralizing Antibody Assay

Heat inactivated sera samples are diluted 1:10 with EMEM and seriallydiluted further 1:4. Diluted serum samples are incubated with PIV3(25-50 pfu) for 1 hour at room temperature and inoculated in duplicatesonto confluent MA-104 monolayers in 24-well plates. After two-hourincubation at 37° C. in a 5% CO₂ incubator, the wells are overlaid with0.75% Methylcellulose medium. After 4 days of incubation, the overlaysare removed and the cells are fixed and stained with 0.1% crystal violetfor one hour and then rinsed and air dried. The corresponding reciprocalneutralizing antibody titers are determined at the 60% reductionend-point of the virus control using a statistics program. Neutralizingtiters are reported as mean+/−SD of log 2 transformed titers for allanimals in a group at a given time point.

hMPV or PIV3 neutralizing antibody titers could be detected in allpreviously-exposed AGM (Groups 11-13 for hMPV and Groups 14-16 for PIV3)and were stable for the 4 weeks preceding immunization (FIGS. 15A-15B).A single 200 μg dose of the hMPV-F/PIV3-F/PIV3-HN mRNA vaccine boostedneutralizing antibody titers to both hMPV (Group 12) and PIV3 (Group 15)by approximately 10 fold. A single 50 μg dose boosted neutralizingantibody titers to hMPV by approximately 8-fold (Groups 13) and to PIV3by approximately 4-fold (Group 16). In all cases the peak neutralizingantibody response was reached by 14 days post immunization, and wasgenerally stable for the subsequent 42 days.

These data show that the hMPV/PIV3 combination vaccine is a potentbooster of neutralizing antibodies primed by hMPV or PIV3 infection inAfrican Green Monkeys.

Example 17 Immunogenicity and Efficacy of hMPV/PIV3 mRNA Vaccines inAfrican Green Monkeys

Lipid nanoparticle (LNP)-formulated combinations of mRNA encoding thefollowing antigens:

-   -   hMPV Fusion (F) protein (Strain: A/TN92-4) (SEQ ID NO: 4)    -   PIV3 F protein (strain: PER/FLA4815/2008) (SEQ ID NO: 5)    -   PIV3 hemagglutinin-neuraminidase (HN) protein        (Strain: 612507167) (SEQ IDNO: 6)

The mRNAs were formulated in a mixture of 4 lipids, including Compound1; 1,2-dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG2000-DMG);1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and cholesterol.

The combination vaccine consists of hMPV-F and PIV3-F mRNA co-formulatedat a 1:1 mass ratio in LNP.

The immunogenicity and efficacy of hMPV/PIV3 mRNA vaccines was evaluatedin the African Green Monkey models of hMPV or PIV3 challenge. Groups ofthree African Green Monkeys were immunized with different dose levels ofLNP-formulated mRNAs encoding hMPV-F, PIV3-F, and/or PIV3 HN asindicated in Table 6. Control groups were inoculated withphosphate-buffered saline (PBS). All animals were immunizedintramuscularly (IM) on Days 0 and 28. Serum was collected before thefirst immunization and on Days 27 and 56 for measurement of neutralizingantibody titers to hMPV (Groups 1-4) or PIV3 (Groups 5-10) by 60% plaquereduction neutralization test (PRNT). Animals in groups 1-4 wereinoculated intratracheally on Day 57 with 5×10⁵ plaque-forming units(pfu) of hMPV strain NL/1/00(A1) and viral load was determined by plaqueassay on nose and lung samples collected on Day 61 (4 days postchallenge). Animals in groups 5-10 were inoculated intra-nasally andintratracheally on Day 57 with 1×10⁶ pfu of PIV3 strain JS and viralload was determined by plaque assay on nose and lung samples collectedon Day 60 (3 days post challenge).

TABLE 6 mRNA dose (μg) Vaccine Challenge Group n Vaccine total hMPV-FPIV3-F PIV3-HN Schedule (Day 57) 1 3 hMPV-F/PIV3-F/PIV3-HN 200 100 50 50Day 0 & 28 hMPV 2 3 hMPV-F/PIV3-F/PIV3-HN 100 50 25 25 Day 0 & 28 hMPV 33 hMPV-F/PIV3-F/PIV3-HN  10 5 2.5 2.5 Day 0 & 28 hMPV 4 3 PBS NA NA NANA Day 0 & 28 hMPV 5 3 hMPV-F/PIV3-F/PIV3-HN 200 100 50 50 Day 0 & 28PIV3 6 3 hMPV-F/PIV3-F/PIV3-HN 100 50 25 25 Day 0 & 28 PIV3 7 3hMPV-F/PIV3-F/PIV3-HN  10 5 2.5 2.5 Day 0 & 28 PIV3 8 3 hMPV-F/PIV3-F100 50 50 0 Day 0 & 28 PIV3 9 3 hMPV-F/PIV3-HN 100 50 0 50 Day 0 & 28PIV3 10 3 PBS NA NA NA NA Day 0 & 28 PIV3 NA—not applicablehMPV Lung and Nose Viral Titration

Lung and nose homogenates are clarified by centrifugation and diluted inEMEM. Confluent MK-2 monolayers are infected in duplicates with dilutedhomogenates in 24 well plates. After one hour incubation at 37° C. in a5% CO₂ incubator, the wells are overlaid with 0.75% methylcellulosemedium. After 7 days of incubation, the overlays are removed and thecells are fixed for one hour and air dried for immune-staining. Uponblocking the wells, mouse anti-hMPV nucoleoprotein (N) at a 1:1000dilution to each well, followed by horseradish peroxidase (HRP)conjugated goat anti-mouse IgG diluted at 1:1000. TrueBlue peroxidasesubstrate is added to each well and incubated at room temperature for 10to 15 minutes. Visible plaques are counted and virus titers areexpressed as pfu per gram (g) of tissue. Viral titers are reported asmean±standard deviation (SD) of log 10 transformed values for allanimals in a group.

PIV3 Lung and Nose Viral Titration

Lung and nose homogenates are clarified by centrifugation and diluted inEMEM. Confluent MA-104 (monkey kidney cells) monolayers are infected induplicates with diluted homogenates in 24 well plates. After two hourincubation at 37° C. in a 5% CO₂ incubator, the wells are overlaid with0.75% methylcellulose medium. After 4 days of incubation, the overlaysare removed and the cells are fixed and stained with 0.1% crystal violetfor one hour and then rinsed and air dried. Plaques are counted andvirus titers are expressed as pfu/g of tissue. Viral loads are reportedas mean+/−SD of log 10 transformed values for all animals in a group.

hMPV neutralizing Antibody Assay

Heat inactivated sera samples are diluted 1:10 with OptiMEM and seriallydiluted further 1:4. Diluted serum samples are incubated with hMPV/A2(25-50 pfu) for 1 hour at room temperature and inoculated in duplicatesonto confluent MK-2 monolayers in 24-well plates. After one hourincubation at 37° C. in a 5% CO₂ incubator, the wells are overlaid with0.75% Methylcellulose medium. After 7 days of incubation, the overlaysare removed and washed once in PBS. The cells are fixed in coldacetone/methanol solution for one hour and air dried forimmuno-staining. The cells are permeablized in 0.4% Triton-X solutionand incubated in blocking solution (10% bovine serum albumin). Mouseanti-hMPV N protein at a 1:1,000 dilution is added to each well,followed by HRP conjugated rabbit anti-mouse IgG diluted at 1:5,000. AECchromogen detection solution is used for coloration after two hours ofincubation or until red plaques are visible. Plaques are counted andvirus titers are expressed as plaque forming units. The correspondingreciprocal neutralizing antibody titers are determined at the 60%reduction end-point of the virus control using a statistics program“plqrd.manual.entry”. Neutralizing titers are reported as mean+/−SD oflog 2 transformed titers for all animals in a group at a given timepoint.

PIV3 Neutralizing Antibody Assay

Heat inactivated sera samples are diluted 1:10 with EMEM and seriallydiluted further 1:4. Diluted serum samples are incubated with PIV3(25-50 pfu) for 1 hour at room temperature and inoculated in duplicatesonto confluent MA-104 monolayers in 24 well plates. After two hourincubation at 37° C. in a 5% CO₂ incubator, the wells are overlaid with0.75% Methylcellulose medium. After 4 days of incubation, the overlaysare removed and the cells are fixed and stained with 0.1% crystal violetfor one hour and then rinsed and air dried. The corresponding reciprocalneutralizing antibody titers are determined at the 60% reductionend-point of the virus control using a statistics program. Neutralizingtiters are reported as mean+/−SD of log 2 transformed titers for allanimals in a group at a given time point.

Results

hMPV neutralizing antibodies were detected in serum of the majority ofanimals 28 days after the first immunization with thehMPV-F/PIV3-F/PIV3-HN mRNA vaccine, and titers were boosted by thesecond immunization (FIG. 16A). The magnitude of the responses werequite high in animals dosed with 200 or 100 μg mRNA (Groups 1 and 2;containing 100 and 50 μg of hMPV-F mRNA, respectively), but wassignificantly lower in animals dosed with 10 μg mRNA (Group 3;containing 5 μg of hMPV-F mRNA). PIV3 neutralizing antibodies weredetected in serum of the majority of animals 28 days after the firstimmunization with the hMPV/PIV3 mRNA vaccines, and titers were boostedby the second immunization (FIG. 16B). The magnitude of the responseswere quite high in all groups, although dose dependent (Groups 5-7). Theresponse induced by the PIV3-F mRNA was equivalent or greater to theresponse induced by the PIV3-HN mRNA (Group 8 vs. Group 9), and addingPIV3-HN to PIV-F did not enhance PIV3 neutralizing antibody titers(Group 8 vs. Group 5).

The ability of the hMPV/PIV3 mRNA vaccines to protect African GreenMonkeys against challenge was determined by measuring viral load inlungs and noses from immunized animals 4 days after hMPV intratrachealinoculation (Groups 1-4) or 3 days after PIV3 intratracheal andintranasal inoculation (Groups 5-10) (FIGS. 17A-17B). High levels ofboth viruses were detected in PBS control animals (Groups 4 and 10), butwere below the limit of quantification in animals immunized with 100 μgor greater mRNA (Groups 1, 2, 5, 6, 8 and 9), demonstrating that thehMPV/PIV3 combination vaccine affords full protection against bothviruses in the lung and the nose. The 10 μg low dose vaccine (Groups 3and 7) afforded some, but not complete protection against hMPV and PIV3.The animals immunized with the hMPV-F/PIV3-F vaccine (Group 8, whichdoes not include PIV3-HN) were also protected from PIV3 challenge,demonstrating that the immune response to the PIV3-F protein issufficient for protection against PIV3.

It should be understood that any of the mRNA sequences described hereinmay include a 5′ UTR and/or a 3′ UTR. The UTR sequences may be selectedfrom the following sequences, or other known UTR sequences may be used.It should also be understood that any of the mRNA constructs describedherein may further comprise a polyA tail and/or cap (e.g.,7mG(5′)ppp(5′)NlmpNp). Further, while many of the mRNAs and encodedantigen sequences described herein include a signal peptide and/or apeptide tag (e.g., C-terminal His tag), it should be understood that theindicated signal peptide and/or peptide tag may be substituted for adifferent signal peptide and/or peptide tag, or the signal peptideand/or peptide tag may be omitted.

5′ UTR: (SEQ ID NO: 12) GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC3′ UTR: (SEQ ID NO: 13)UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAA UAAAGUCUGAGUGGGCGGC

TABLE 7 Sequences of Antigens encoded by hMPV/HPIV3 mRNA vaccineshMPV F proteinSEQ ID NO: 14 consists of from 5′ end to 3′ end, 5′ UTR SEQ ID 14NO: 12, mRNA ORF SEQ ID NO: 4, and 3′ UTR SEQ ID NO: 13. Chemistry1-methylpseudouridine Cap 7 mG(5′)ppp(5′)NlmpNp 5′ UTRGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUA 12 AGAGCCACC ORF of DNAATGAGCTGGAAGGTGGTGATTATCTTCAGCCTGCTGATTAC 1 ConstructACCTCAACACGGCCTGAAGGAGAGCTACCTGGAAGAGAGCTGCTCCACCATCACCGAGGGCTACCTGAGCGTGCTGCGGACCGGCTGGTACACCAACGTGTTCACCCTGGAGGTGGGCGACGTGGAGAACCTGACCTGCAGCGACGGCCCTAGCCTGATCAAGACCGAGCTGGACCTGACCAAGAGCGCTCTGAGAGAGCTGAAGACCGTGTCCGCCGACCAGCTGGCCAGAGAGGAACAGATCGAGAACCCTCGGCAGAGCAGATTCGTGCTGGGCGCCATCGCTCTGGGAGTCGCCGCTGCCGCTGCAGTGACAGCTGGAGTGGCCATTGCTAAGACCATCAGACTGGAAAGCGAGGTGACAGCCATCAACAATGCCCTGAAGAAGACCAACGAGGCCGTGAGCACCCTGGGCAATGGAGTGAGAGTGCTGGCCACAGCCGTGCGGGAGCTGAAGGACTTCGTGAGCAAGAACCTGACCAGAGCCATCAACAAGAACAAGTGCGACATCGATGACCTGAAGATGGCCGTGAGCTTCTCCCAGTTCAACAGACGGTTCCTGAACGTGGTGAGACAGTTCTCCGACAACGCTGGAATCACACCTGCCATTAGCCTGGACCTGATGACCGACGCCGAGCTGGCTAGAGCCGTGCCCAACATGCCCACCAGCGCTGGCCAGATCAAGCTGATGCTGGAGAACAGAGCCATGGTGCGGAGAAAGGGCTTCGGCATCCTGATTGGGGTGTATGGAAGCTCCGTGATCTACATGGTGCAGCTGCCCATCTTCGGCGTGATCGACACACCCTGCTGGATCGTGAAGGCCGCTCCTAGCTGCTCCGAGAAGAAAGGAAACTATGCCTGTCTGCTGAGAGAGGACCAGGGCTGGTACTGCCAGAACGCCGGAAGCACAGTGTACTATCCCAACGAGAAGGACTGCGAGACCAGAGGCGACCACGTGTTCTGCGACACCGCTGCCGGAATCAACGTGGCCGAGCAGAGCAAGGAGTGCAACATCAACATCAGCACAACCAACTACCCCTGCAAGGTGAGCACCGGACGGCACCCCATCAGCATGGTGGCTCTGAGCCCTCTGGGCGCTCTGGTGGCCTGCTATAAGGGCGTGTCCTGTAGCATCGGCAGCAATCGGGTGGGCATCATCAAGCAGCTGAACAAGGGATGCTCCTACATCACCAACCAGGACGCCGACACCGTGACCATCGACAACACCGTGTACCAGCTGAGCAAGGTGGAGGGCGAGCAGCACGTGATCAAGGGCAGACCCGTGAGCTCCAGCTTCGACCCCATCAAGTTCCCTGAGGACCAGTTCAACGTGGCCCTGGACCAGGTGTTTGAGAACATCGAGAACAGCCAGGCCCTGGTGGACCAGAGCAACAGAATCCTGTCCAGCGCTGAGAAGGGCAACACCGGCTTCATCATTGTGATCATTCTGATCGCCGTGCTGGGCAGCTCCATGATCCTGGTGAGCATCTTCATCATTATCAAGAAGACCAAGAAACCCACCGGAGCCCCTCCTGAGCTGAGCGGCGTGACCAACAATGGCTTCATTCCCC ACAACTGA ORF of mRNAAUGAGCUGGAAGGUGGUGAUUAUCUUCAGCCUGCUGAUU 4 ConstructACACCUCAACACGGCCUGAAGGAGAGCUACCUGGAAGAGAGCUGCUCCACCAUCACCGAGGGCUACCUGAGCGUGCUGCGGACCGGCUGGUACACCAACGUGUUCACCCUGGAGGUGGGCGACGUGGAGAACCUGACCUGCAGCGACGGCCCUAGCCUGAUCAAGACCGAGCUGGACCUGACCAAGAGCGCUCUGAGAGAGCUGAAGACCGUGUCCGCCGACCAGCUGGCCAGAGAGGAACAGAUCGAGAACCCUCGGCAGAGCAGAUUCGUGCUGGGCGCCAUCGCUCUGGGAGUCGCCGCUGCCGCUGCAGUGACAGCUGGAGUGGCCAUUGCUAAGACCAUCAGACUGGAAAGCGAGGUGACAGCCAUCAACAAUGCCCUGAAGAAGACCAACGAGGCCGUGAGCACCCUGGGCAAUGGAGUGAGAGUGCUGGCCACAGCCGUGCGGGAGCUGAAGGACUUCGUGAGCAAGAACCUGACCAGAGCCAUCAACAAGAACAAGUGCGACAUCGAUGACCUGAAGAUGGCCGUGAGCUUCUCCCAGUUCAACAGACGGUUCCUGAACGUGGUGAGACAGUUCUCCGACAACGCUGGAAUCACACCUGCCAUUAGCCUGGACCUGAUGACCGACGCCGAGCUGGCUAGAGCCGUGCCCAACAUGCCCACCAGCGCUGGCCAGAUCAAGCUGAUGCUGGAGAACAGAGCCAUGGUGCGGAGAAAGGGCUUCGGCAUCCUGAUUGGGGUGUAUGGAAGCUCCGUGAUCUACAUGGUGCAGCUGCCCAUCUUCGGCGUGAUCGACACACCCUGCUGGAUCGUGAAGGCCGCUCCUAGCUGCUCCGAGAAGAAAGGAAACUAUGCCUGUCUGCUGAGAGAGGACCAGGGCUGGUACUGCCAGAACGCCGGAAGCACAGUGUACUAUCCCAACGAGAAGGACUGCGAGACCAGAGGCGACCACGUGUUCUGCGACACCGCUGCCGGAAUCAACGUGGCCGAGCAGAGCAAGGAGUGCAACAUCAACAUCAGCACAACCAACUACCCCUGCAAGGUGAGCACCGGACGGCACCCCAUCAGCAUGGUGGCUCUGAGCCCUCUGGGCGCUCUGGUGGCCUGCUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAAUCGGGUGGGCAUCAUCAAGCAGCUGAACAAGGGAUGCUCCUACAUCACCAACCAGGACGCCGACACCGUGACCAUCGACAACACCGUGUACCAGCUGAGCAAGGUGGAGGGCGAGCAGCACGUGAUCAAGGGCAGACCCGUGAGCUCCAGCUUCGACCCCAUCAAGUUCCCUGAGGACCAGUUCAACGUGGCCCUGGACCAGGUGUUUGAGAACAUCGAGAACAGCCAGGCCCUGGUGGACCAGAGCAACAGAAUCCUGUCCAGCGCUGAGAAGGGCAACACCGGCUUCAUCAUUGUGAUCAUUCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGAGCAUCUUCAUCAUUAUCAAGAAGACCAAGAAACCCACCGGAGCCCCUCCUGAGCUGAGCGGCGUGACCAACAAUGGCUUC AUUCCCCACAACUGA 3′UTRUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCC 13CCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGG C CorrespondingMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWY 7 amino acidTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALRELKTVSAD sequenceQLAREEQIENPRQSRFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTK KPTGAPPELSGVTNNGFIPHNPolyA tail 100 nt hPIV3 F proteinSEQ ID NO: 15 consists of from 5′ end to 3′ end, 5′ UTR SEQ ID 15NO: 12, mRNA ORF SEQ ID NO: 5, and 3′ UTR SEQ ID NO: 13. Chemistry1-methylpseudouridine Cap 7  mG(5′)ppp(5′)NlmpNp 5′ UTRGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUA 12 AGAGCCACC ORF of DNAATGCCCATCAGCATCCTGCTGATCATCACCACAATGATCAT 2 ConstructGGCCAGCCACTGCCAGATCGACATCACCAAGCTGCAGCACGTGGGCGTGCTCGTGAACAGCCCCAAGGGCATGAAGATCAGCCAGAACTTCGAGACACGCTACCTGATCCTGAGCCTGATCCCCAAGATCGAGGACAGCAACAGCTGCGGCGACCAGCAGATCAAGCAGTACAAGCGGCTGCTGGACAGACTGATCATCCCCCTGTACGACGGCCTGCGGCTGCAGAAAGACGTGATCGTGACCAACCAGGAAAGCAACGAGAACACCGACCCCCGGACCGAGAGATTCTTCGGCGGCGTGATCGGCACAATCGCCCTGGGAGTGGCCACAAGCGCCCAGATTACAGCCGCTGTGGCCCTGGTGGAAGCCAAGCAGGCCAGAAGCGACATCGAGAAGCTGAAAGAGGCCATCCGGGACACCAACAAGGCCGTGCAGAGCGTGCAGTCCAGCGTGGGCAATCTGATCGTGGCCATCAAGTCCGTGCAGGACTACGTGAACAAAGAAATCGTGCCCTCTATCGCCCGGCTGGGCTGTGAAGCTGCCGGACTGCAGCTGGGCATTGCCCTGACACAGCACTACAGCGAGCTGACCAACATCTTCGGCGACAACATCGGCAGCCTGCAGGAAAAGGGCATTAAGCTGCAGGGAATCGCCAGCCTGTACCGCACCAACATCACCGAGATCTTCACCACCAGCACCGTGGATAAGTACGACATCTACGACCTGCTGTTCACCGAGAGCATCAAAGTGCGCGTGATCGACGTGGACCTGAACGACTACAGCATCACCCTGCAAGTGCGGCTGCCCCTGCTGACCAGACTGCTGAACACCCAGATCTACAAGGTGGACAGCATCTCCTACAACATCCAGAACCGCGAGTGGTACATCCCTCTGCCCAGCCACATTATGACCAAGGGCGCCTTTCTGGGCGGAGCCGACGTGAAAGAGTGCATCGAGGCCTTCAGCAGCTACATCTGCCCCAGCGACCCTGGCTTCGTGCTGAACCACGAGATGGAAAGCTGCCTGAGCGGCAACATCAGCCAGTGCCCCAGAACCACCGTGACCTCCGACATCGTGCCCAGATACGCCTTCGTGAATGGCGGCGTGGTGGCCAACTGCATCACCACCACCTGTACCTGCAACGGCATCGGCAACCGGATCAACCAGCCTCCCGATCAGGGCGTGAAGATTATCACCCACAAAGAGTGTAACACCATCGGCATCAACGGCATGCTGTTCAATACCAACAAAGAGGGCACCCTGGCCTTCTACACCCCCGACGATATCACCCTGAACAACTCCGTGGCTCTGGACCCCATCGACATCTCCATCGAGCTGAACAAGGCCAAGAGCGACCTGGAAGAGTCCAAAGAGTGGATCCGGCGGAGCAACCAGAAGCTGGACTCTATCGGCAGCTGGCACCAGAGCAGCACCACCATCATCGTGATCCTGATTATGATGATTATCCTGTTCATCATCAACATTACCATCATCACTATCGCCATTAAGTACTACCGGATCCAGAAACGGAACCGGGTGGACCAGAATGACAAGCCCTACGTGCTGACAAACAAG ORF of mRNAAUGCCCAUCAGCAUCCUGCUGAUCAUCACCACAAUGAUC 5 ConstructAUGGCCAGCCACUGCCAGAUCGACAUCACCAAGCUGCAGCACGUGGGCGUGCUCGUGAACAGCCCCAAGGGCAUGAAGAUCAGCCAGAACUUCGAGACACGCUACCUGAUCCUGAGCCUGAUCCCCAAGAUCGAGGACAGCAACAGCUGCGGCGACCAGCAGAUCAAGCAGUACAAGCGGCUGCUGGACAGACUGAUCAUCCCCCUGUACGACGGCCUGCGGCUGCAGAAAGACGUGAUCGUGACCAACCAGGAAAGCAACGAGAACACCGACCCCCGGACCGAGAGAUUCUUCGGCGGCGUGAUCGGCACAAUCGCCCUGGGAGUGGCCACAAGCGCCCAGAUUACAGCCGCUGUGGCCCUGGUGGAAGCCAAGCAGGCCAGAAGCGACAUCGAGAAGCUGAAAGAGGCCAUCCGGGACACCAACAAGGCCGUGCAGAGCGUGCAGUCCAGCGUGGGCAAUCUGAUCGUGGCCAUCAAGUCCGUGCAGGACUACGUGAACAAAGAAAUCGUGCCCUCUAUCGCCCGGCUGGGCUGUGAAGCUGCCGGACUGCAGCUGGGCAUUGCCCUGACACAGCACUACAGCGAGCUGACCAACAUCUUCGGCGACAACAUCGGCAGCCUGCAGGAAAAGGGCAUUAAGCUGCAGGGAAUCGCCAGCCUGUACCGCACCAACAUCACCGAGAUCUUCACCACCAGCACCGUGGAUAAGUACGACAUCUACGACCUGCUGUUCACCGAGAGCAUCAAAGUGCGCGUGAUCGACGUGGACCUGAACGACUACAGCAUCACCCUGCAAGUGCGGCUGCCCCUGCUGACCAGACUGCUGAACACCCAGAUCUACAAGGUGGACAGCAUCUCCUACAACAUCCAGAACCGCGAGUGGUACAUCCCUCUGCCCAGCCACAUUAUGACCAAGGGCGCCUUUCUGGGCGGAGCCGACGUGAAAGAGUGCAUCGAGGCCUUCAGCAGCUACAUCUGCCCCAGCGACCCUGGCUUCGUGCUGAACCACGAGAUGGAAAGCUGCCUGAGCGGCAACAUCAGCCAGUGCCCCAGAACCACCGUGACCUCCGACAUCGUGCCCAGAUACGCCUUCGUGAAUGGCGGCGUGGUGGCCAACUGCAUCACCACCACCUGUACCUGCAACGGCAUCGGCAACCGGAUCAACCAGCCUCCCGAUCAGGGCGUGAAGAUUAUCACCCACAAAGAGUGUAACACCAUCGGCAUCAACGGCAUGCUGUUCAAUACCAACAAAGAGGGCACCCUGGCCUUCUACACCCCCGACGAUAUCACCCUGAACAACUCCGUGGCUCUGGACCCCAUCGACAUCUCCAUCGAGCUGAACAAGGCCAAGAGCGACCUGGAAGAGUCCAAAGAGUGGAUCCGGCGGAGCAACCAGAAGCUGGACUCUAUCGGCAGCUGGCACCAGAGCAGCACCACCAUCAUCGUGAUCCUGAUUAUGAUGAUUAUCCUGUUCAUCAUCAACAUUACCAUCAUCACUAUCGCCAUUAAGUACUACCGGAUCCAGAAACGGAACCGGGUGGACCAGAAUGACAAGCCCUACGUGCUGAC AAACAAG 3′ UTRUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCC 13CCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGG C CorrespondingMPISILLIITTMIMASHCQIDITKLQHVGVLVNSPKGMKISQNFE 8 amino acidTRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLRLQK sequenceDVIVTNQESNENTDPRTERFFGGVIGTIALGVATSAQITAAVALVEAKQARSDIEKLKEAIRDTNKAVQSVQSSVGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGSLQEKGIKLQGIASLYRTNITEIFTTSTVDKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTTVTSDIVPRYAFVNGGVVANCITTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPDDITLNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGSWHQSSTTIIVILIMMIILFIINITIITIAIKYYRIQKRNRVDQNDKPYV LTNK PolyA tail 100 nthPIV3 HN proteinSEQ ID NO: 16 consists of from 5′ end to 3′ end, 5′ UTR SEQ ID  16NO: 12, mRNA ORF SEQ ID NO: 6, and 3′ UTR SEQ ID NO: 13. Chemistry1-methylpseudouridine Cap 7 mG(5′)ppp(5′)NlmpNp 5′ UTRGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUA 12 AGAGCCACC ORF of DNAATGGAATACTGGAAGCACACCAACCACGGCAAGGACGCCG 3 ConstructGCAACGAGCTGGAAACCAGCACAGCCACACACGGCAACAAGCTGACCAACAAGATCACCTACATCCTGTGGACCATCACCCTGGTGCTGCTGAGCATCGTGTTCATCATCGTGCTGACCAATAGCATCAAGAGCGAGAAGGCCAGAGAGAGCCTGCTGCAGGACATCAACAACGAGTTCATGGAAGTGACCGAGAAGATCCAGGTGGCCAGCGACAACACCAACGACCTGATCCAGAGCGGCGTGAACACCCGGCTGCTGACCATCCAGAGCCACGTGCAGAACTACATCCCCATCAGCCTGACCCAGCAGATCAGCGACCTGCGGAAGTTCATCAGCGAGATCACCATCCGGAACGACAACCAGGAAGTGCCCCCCCAGAGAATCACCCACGACGTGGGCATCAAGCCCCTGAACCCCGACGATTTCTGGCGGTGTACAAGCGGCCTGCCCAGCCTGATGAAGACCCCCAAGATCCGGCTGATGCCTGGCCCTGGACTGCTGGCCATGCCTACCACAGTGGATGGCTGTGTGCGGACCCCCAGCCTCGTGATCAACGATCTGATCTACGCCTACACCAGCAACCTGATCACCCGGGGCTGCCAGGATATCGGCAAGAGCTACCAGGTGCTGCAGATCGGCATCATCACCGTGAACTCCGACCTGGTGCCCGACCTGAACCCTCGGATCAGCCACACCTTCAACATCAACGACAACAGAAAGAGCTGCAGCCTGGCTCTGCTGAACACCGACGTGTACCAGCTGTGCAGCACCCCCAAGGTGGACGAGAGAAGCGACTACGCCAGCAGCGGCATCGAGGATATCGTGCTGGACATCGTGAACTACGACGGCAGCATCAGCACCACCCGGTTCAAGAACAACAACATCAGCTTCGACCAGCCCTACGCCGCCCTGTACCCTTCTGTGGGCCCTGGCATCTACTACAAGGGCAAGATCATCTTCCTGGGCTACGGCGGCCTGGAACACCCCATCAACGAGAACGCCATCTGCAACACCACCGGCTGCCCTGGCAAGACCCAGAGAGACTGCAATCAGGCCAGCCACAGCCCCTGGTTCAGCGACCGCAGAATGGTCAACTCTATCATCGTGGTGGACAAGGGCCTGAACAGCGTGCCCAAGCTGAAAGTGTGGACAATCAGCATGCGCCAGAACTACTGGGGCAGCGAGGGCAGACTTCTGCTGCTGGGAAACAAGATCTACATCTACACCCGGTCCACCAGCTGGCACAGCAAACTGCAGCTGGGAATCATCGACATCACCGACTACAGCGACATCCGGATCAAGTGGACCTGGCACAACGTGCTGAGCAGACCCGGCAACAATGAGTGCCCTTGGGGCCACAGCTGCCCCGATGGATGTATCACCGGCGTGTACACCGACGCCTACCCCCTGAATCCTACCGGCTCCATCGTGTCCAGCGTGATCCTGGACAGCCAGAAAAGCAGAGTGAACCCCGTGATCACATACAGCACCGCCACCGAGAGAGTGAACGAACTGGCCATCAGAAACAAGACCCTGAGCGCCGGCTACACCACCACAAGCTGCATCACACACTACAACAAGGGCTACTGCTTCCACATCGTGGAAATCAACCACAAGTCCCTGAACACCTTCCAGCCCATGCTGTTCAAGACC GAGATCCCCAAGAGCTGCTCCORF of mRNA AUGGAAUACUGGAAGCACACCAACCACGGCAAGGACGCC 6 ConstructGGCAACGAGCUGGAAACCAGCACAGCCACACACGGCAACAAGCUGACCAACAAGAUCACCUACAUCCUGUGGACCAUCACCCUGGUGCUGCUGAGCAUCGUGUUCAUCAUCGUGCUGACCAAUAGCAUCAAGAGCGAGAAGGCCAGAGAGAGCCUGCUGCAGGACAUCAACAACGAGUUCAUGGAAGUGACCGAGAAGAUCCAGGUGGCCAGCGACAACACCAACGACCUGAUCCAGAGCGGCGUGAACACCCGGCUGCUGACCAUCCAGAGCCACGUGCAGAACUACAUCCCCAUCAGCCUGACCCAGCAGAUCAGCGACCUGCGGAAGUUCAUCAGCGAGAUCACCAUCCGGAACGACAACCAGGAAGUGCCCCCCCAGAGAAUCACCCACGACGUGGGCAUCAAGCCCCUGAACCCCGACGAUUUCUGGCGGUGUACAAGCGGCCUGCCCAGCCUGAUGAAGACCCCCAAGAUCCGGCUGAUGCCUGGCCCUGGACUGCUGGCCAUGCCUACCACAGUGGAUGGCUGUGUGCGGACCCCCAGCCUCGUGAUCAACGAUCUGAUCUACGCCUACACCAGCAACCUGAUCACCCGGGGCUGCCAGGAUAUCGGCAAGAGCUACCAGGUGCUGCAGAUCGGCAUCAUCACCGUGAACUCCGACCUGGUGCCCGACCUGAACCCUCGGAUCAGCCACACCUUCAACAUCAACGACAACAGAAAGAGCUGCAGCCUGGCUCUGCUGAACACCGACGUGUACCAGCUGUGCAGCACCCCCAAGGUGGACGAGAGAAGCGACUACGCCAGCAGCGGCAUCGAGGAUAUCGUGCUGGACAUCGUGAACUACGACGGCAGCAUCAGCACCACCCGGUUCAAGAACAACAACAUCAGCUUCGACCAGCCCUACGCCGCCCUGUACCCUUCUGUGGGCCCUGGCAUCUACUACAAGGGCAAGAUCAUCUUCCUGGGCUACGGCGGCCUGGAACACCCCAUCAACGAGAACGCCAUCUGCAACACCACCGGCUGCCCUGGCAAGACCCAGAGAGACUGCAAUCAGGCCAGCCACAGCCCCUGGUUCAGCGACCGCAGAAUGGUCAACUCUAUCAUCGUGGUGGACAAGGGCCUGAACAGCGUGCCCAAGCUGAAAGUGUGGACAAUCAGCAUGCGCCAGAACUACUGGGGCAGCGAGGGCAGACUUCUGCUGCUGGGAAACAAGAUCUACAUCUACACCCGGUCCACCAGCUGGCACAGCAAACUGCAGCUGGGAAUCAUCGACAUCACCGACUACAGCGACAUCCGGAUCAAGUGGACCUGGCACAACGUGCUGAGCAGACCCGGCAACAAUGAGUGCCCUUGGGGCCACAGCUGCCCCGAUGGAUGUAUCACCGGCGUGUACACCGACGCCUACCCCCUGAAUCCUACCGGCUCCAUCGUGUCCAGCGUGAUCCUGGACAGCCAGAAAAGCAGAGUGAACCCCGUGAUCACAUACAGCACCGCCACCGAGAGAGUGAACGAACUGGCCAUCAGAAACAAGACCCUGAGCGCCGGCUACACCACCACAAGCUGCAUCACACACUACAACAAGGGCUACUGCUUCCACAUCGUGGAAAUCAACCACAAGUCCCUGAACACCUUCCAGCCCAUGCUGUUCAA GACCGAGAUCCCCAAGAGCUGCUCC3′ UTR UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCC 13CCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGG C CorrespondingMEYWKHTNHGKDAGNELETSTATHGNKLTNKITYILWTITLV 9 amino acidLLSIVFIIVLTNSIKSEKARESLLQDINNEFMEVTEKIQVASDNT sequenceNDLIQSGVNTRLLTIQSHVQNYIPISLTQQISDLRKFISEITIRNDNQEVPPQRITHDVGIKPLNPDDFWRCTSGLPSLMKTPKIRLMPGPGLLAMPTTVDGCVRTPSLVINDLIYAYTSNLITRGCQDIGKSYQVLQIGIITVNSDLVPDLNPRISHTFNINDNRKSCSLALLNTDVYQLCSTPKVDERSDYASSGIEDIVLDIVNYDGSISTTRFKNNNISFDQPYAALYPSVGPGIYYKGKIIFLGYGGLEHPINENAICNTTGCPGKTQRDCNQASHSPWFSDRRMVNSIIVVDKGLNSVPKLKVWTISMRQNYWGSEGRLLLLGNKIYIYTRSTSWHSKLQLGIIDITDYSDIRIKWTWHNVLSRPGNNECPWGHSCPDGCITGVYTDAYPLNPTGSIVSSVILDSQKSRVNPVITYSTATERVNELAIRNKTLSAGYTTTSCITHYNKGYCFHIVEINHKSLNTFQPMLFKTEIP KSCS PolyA tail 100 nt

International Patent Application No. PCT/US2016/58327 is hereinincorporated by reference in its entirety.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A vaccine comprising (a) a RNA polynucleotidecomprising the nucleic acid sequence identified by SEQ ID NO:4 or a RNApolynucleotide comprising a nucleic acid sequence at least 95% identicalto the nucleic acid sequence identified by SEQ ID NO:4 encoding a humanmetapneumovirus (hMPV) F protein, and (b) a RNA polynucleotidecomprising the nucleic acid sequence identified by SEQ ID NO:5 or a RNApolynucleotide comprising a nucleic acid sequence at least 95% identicalto the nucleic acid sequence identified by SEQ ID NO:5 encoding a humanparainfluenza virus 3 (hPIV3) F protein.
 2. The vaccine of claim 1,wherein the vaccine includes a 5′ UTR, a 3′ UTR, a polyA tail (e.g., 100nucleotides), a cap (e.g., 7mG(5′)ppp(5′)NlmpNp), or any combination oftwo or more of the foregoing components.
 3. The vaccine of claim 2, wherein the 5′ UTR comprises a sequence identified by SEQ ID NO:12. 4.The vaccine of claim 2 or 3, wherein the 3′ UTR comprises a sequenceidentified by SEQ ID NO:13.
 5. The vaccine of any one of claim 1-4,wherein the RNA polynucleotide of (a) and/or (b) is chemically modified.6. The vaccine of claim 5, wherein the RNA polynucleotide of (a) and/or(b) comprises 1-methylpseudouridine modifications).
 7. The vaccine ofany one of claims 1-6, wherein the RNA polynucleotide of (a) comprisesthe nucleic acid sequence identified by SEQ ID NO:14 encoding a hMPV Fprotein and the RNA polynucleotide of (b) comprises the nucleic acidsequence identified by SEQ ID NO:15 encoding a hPIV3 F protein.
 8. Thevaccine of any one of claims 1-7 formulated in a cationic lipidnanoparticle.
 9. The vaccine of claim 8, wherein the cationic lipidnanoparticle comprises a mixture of: Compound 1 lipids;1,2-dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG2000-DMG);1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and cholesterol. 10.The vaccine of any one of claims 1-9, wherein the vaccine comprises a12.5 μg-200 12.5 μg dose of the RNA polynucleotide of (a) and a 12.5μg-200 12.5 μg dose of the RNA polynucleotide of (b).
 11. A vaccinecomprising (a) a RNA polynucleotide comprising a the nucleic acidsequence identified by SEQ ID NO:14 encoding a hMPV F protein and (b) aRNA polynucleotide comprising the nucleic acid sequence identified bySEQ ID NO:15 encoding a hPIV3 F protein, wherein the RNA polynucleotideof (a) and the RNA polynucleotide of (b) are co-formulated in a cationiclipid nanoparticle that comprises a mixture of: Compound 1 lipids;1,2-dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG2000-DMG);1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and cholesterol. 12.A vaccine comprising (a) a RNA polynucleotide comprising a the nucleicacid sequence identified by SEQ ID NO:14 encoding a hMPV F protein and(b) a RNA polynucleotide comprising the nucleic acid sequence identifiedby SEQ ID NO:15 encoding a hPIV3 F protein, wherein the RNApolynucleotide of (a) and the RNA polynucleotide of (b) are separatelyformulated in cationic lipid nanoparticles that comprises a mixture of:Compound 1 lipids; 1,2-dimyristoyl-sn-glycerol,methoxypolyethyleneglycol (PEG2000-DMG);1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and cholesterol. 13.The vaccine of claim 11 or 12, wherein the vaccine comprises a 12.5μg-200 12.5 μg dose of the RNA polynucleotide of (a) and a 12.5 μg-20012.5 μg dose of the RNA polynucleotide of (b).
 14. The vaccine of anyone of claims 1-13, wherein hMPV and/or hPIV3 viral load is undetectablein subjects after challenge with the virus(es) following administrationof less than three doses of the vaccine.
 15. The vaccine of claim 14,wherein hMPV and/or hPIV3 viral load is undetectable in subjects afterchallenge with the virus(es) following administration of two doses ofthe vaccine.
 16. The vaccine of claim 15, wherein hMPV and/or hPIV3viral load is undetectable in subjects after challenge with thevirus(es) following administration of a single dose of the vaccine. 17.The vaccine of any one of claims 1-16 wherein the anti-hPIV3neutralizing antibody titer produced in a subject followingadministration of a dose of the vaccine is at least 3-fold higher thanthe anti-hPIV3 neutralizing antibody titer produced in a subjectfollowing administration of a comparable dose of a vaccine comprisingmRNA encoding hPIV3 HN protein.
 18. The vaccine of any one of claims1-17, wherein the vaccine provides an effective immune response againstboth hMPV and hPIV3.
 19. The vaccine of any one of claims 1-18, whereinthe anti-hPIV3 and/or anti-hMPV neutralizing antibody titer produced incotton rats following administration of the vaccine is at least 9 on alog base 2 scale, as measured at the 60% reduction end point of viruscontrol.
 20. The vaccine of any one of claims 1-19, wherein hMPV viralload in the lung and/or nose is below the limit of quantification insubjects following administration of the vaccine and challenge withhMPV.
 21. The vaccine of any one of claims 1-20, wherein hPIV3 viralload in the lung and/or nose is below the limit of quantification insubjects following administration of the vaccine and challenge with thehPIV3.
 22. The vaccine of any one of claims 1-21, wherein a subjectadministered the vaccine does not exhibit symptoms of vaccine-enhancedrespiratory disease.
 23. The vaccine of any one of claims 1-22, whereinthe neutralizing antibody titer against hMPV in a subject followingadministration of a second dose of the vaccine is increased by 8-10 foldat 14 days post-administration of the second dose, and/or wherein theneutralizing antibody titer against hPIV3 in a subject followingadministration of a second dose of the vaccine is increased by 4-10 foldat 14 days post-administration of the second dose.
 24. A method ofinducing an immune response to hMPV and/or hPIV3 in a subject,comprising administering to the subject the vaccine of any one of claims1-23.
 25. A vaccine, comprising: (a) a ribonucleic acid (RNA)polynucleotide encoding a human metapneumovirus (hMPV) antigenicpolypeptide comprising the amino acid sequence identified by SEQ ID NO:7or an amino acid sequence that is at least 95% identical to the aminoacid sequence identified by SEQ ID NO:7; and (b) a RNA polynucleotideencoding human parainfluenza virus 3 (hPIV3) antigenic polypeptidecomprising the amino acid sequence identified by SEQ ID NO:8 or an aminoacid sequence that is at least 95% identical to the amino acid sequenceidentified by SEQ ID NO:8.
 26. The vaccine of claim 25, wherein the RNApolynucleotides of (a) and (b) are formulated in a lipid nanoparticlecomprising a cationic lipid, a PEG-modified lipid, a sterol and anon-cationic lipid.
 27. The vaccine of claim 25 or 26 further comprisinga RNA polynucleotide encoding a respiratory syncytial virus (RSV)antigenic polypeptide.
 28. The vaccine of any one of claims 26-27,wherein the cationic lipid is an ionizable lipid.
 29. The vaccine of anyone of claims 26-28, wherein the sterol is a cholesterol.
 30. Thevaccine of any one of claims 26-29, wherein the non-cationic lipid is aneutral lipid.
 31. The vaccine of any one of claims 26-30, wherein thecationic lipid comprises a compound of Formula I.
 32. The vaccine ofclaim 31, wherein the compound of Formula I is Compound 1 or
 2. 33. Thevaccine of claim 32, wherein the compound of Formula I is Compound 1.34. The vaccine of any one of claims 26-33, wherein the lipidnanoparticle comprises a molar ratio of about 20-60% cationic lipid,0.5-15% PEG-modified lipid, 25-55% sterol, and 25% non-cationic lipid.35. The vaccine of any one of claims 26-34, wherein the lipidnanoparticle comprises a mixture of four lipids, including Compound 1;1,2-dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG2000-DMG);1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and cholesterol. 36.The vaccine of any one of claims 25-35, wherein the RNA encoding hMPVantigenic polypeptide and the RNA encoding hPIV3 antigenic polypeptideare co-formulated at a 1:1 mass ratio.
 37. The vaccine of any one ofclaims 26-36, wherein the lipid nanoparticle has a polydispersity valueof less than 0.4.
 38. The vaccine of any one of claims 26-35, whereinthe lipid nanoparticle has a net neutral charge at a neutral pH value.39. The vaccine of any one of claims 25-38 formulated in an effectiveamount to prevent or treat a lower respiratory hMPV/hPIV3 infection in asubject.
 40. The vaccine of claim 39, wherein the effective amount is 5μg-100 μg of the RNA polynucleotide encoding hMPV antigenic polypeptideand/or 5 μg-100 μg of the RNA polynucleotide encoding hPIV3 antigenicpolypeptide.
 41. The vaccine of claim 40, wherein the effective amountis 12.5 μg of the RNA polynucleotide encoding hMPV antigenic polypeptideand/or 12.5 μg of the RNA polynucleotide encoding hPIV3 antigenicpolypeptide.
 42. The vaccine of claim 40, wherein the effective amountis 25 μg of the RNA polynucleotide encoding hMPV antigenic polypeptideand/or 25 μg of the RNA polynucleotide encoding hPIV3 antigenicpolypeptide.
 43. The vaccine of claim 40, wherein the effective amountis 50 μg of the RNA polynucleotide encoding hMPV antigenic polypeptideand/or 50 μg of the RNA polynucleotide encoding hPIV3 antigenicpolypeptide.
 44. The vaccine of any one of claims 25-43, wherein the RNApolynucleotide encoding hMPV antigenic polypeptide and/or the RNApolynucleotide encoding hPIV3 antigenic polypeptide comprises at leastone chemical modification.
 45. The vaccine of claim 44, wherein thechemical modification is selected from pseudouridine,N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine,4′-thiouridine, 5-methylcytosine, 5-methyluridine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine.
 46. Thevaccine of claim 44 or 45, wherein the chemical modification is in the5-position of the uracil.
 47. The vaccine of any one of claims 44-46,wherein the chemical modification is a N1-methylpseudouridine orN1-ethylpseudouridine.
 48. The vaccine of any one of claims 44-47,wherein at least 80% of the uracil in the open reading frame have achemical modification.
 49. The vaccine of claim 48, wherein at least 90%of the uracil in the open reading frame have a chemical modification.50. The vaccine of claim 49, wherein 100% of the uracil in the openreading frame have a chemical modification.
 51. The vaccine of any oneof claims 25-50, wherein the RNA polynucleotide encoding hMPV antigenicpolypeptide and/or the RNA polynucleotide encoding hPIV3 antigenicpolypeptide further encodes at least one 5′ terminal cap.
 52. Thevaccine of claim 51, wherein the 5′ terminal cap is7mG(5′)ppp(5′)NlmpNp.
 53. The vaccine of any one of claims 25-52 furthercomprising an adjuvant.
 54. The vaccine of claim 53, wherein theadjuvant is a flagellin protein or peptide.
 55. The vaccine of any oneof claims 25-54, wherein hMPV and/or hPIV3 viral load is undetectable insubjects after challenge with the virus(es) following administration ofless than three doses of the vaccine.
 56. The vaccine of claim 55,wherein hMPV and/or hPIV3 viral load is undetectable in subjects afterchallenge with the virus(es) following administration of two doses ofthe vaccine.
 57. The vaccine of claim 56, wherein hMPV and/or hPIV3viral load is undetectable in subjects after challenge with thevirus(es) following administration of a single dose of the vaccine. 58.The vaccine of any one of claims 25-57, wherein the anti-hPIV3neutralizing antibody titer produced in a subject followingadministration of a dose of the vaccine is at least 3-fold higher thanthe anti-hPIV3 neutralizing antibody titer produced in a subjectfollowing administration of a comparable dose of a vaccine comprisingmRNA encoding hPIV3 HN protein.
 59. The vaccine of any one of claims25-58, wherein the vaccine provides an effective immune response againstboth hMPV and hPIV3.
 60. The vaccine of any one of claims 25-59, whereinthe anti-hPIV3 and/or anti-hMPV neutralizing antibody titer produced incotton rats following administration of the vaccine is at least 9 on alog base 2 scale, as measured at the 60% reduction end point of viruscontrol.
 61. The vaccine of any one of claims 25-60, wherein hMPV viralload in the lung and/or nose is below the limit of quantification insubjects following administration of the vaccine and challenge withhMPV.
 62. The vaccine of any one of claims 25-61, wherein hPIV3 viralload in the lung and/or nose is below the limit of quantification insubjects following administration of the vaccine and challenge with thehPIV3.
 63. The vaccine of any one of claims 25-62, wherein a subjectadministered the vaccine does not exhibit symptoms of vaccine-enhancedrespiratory disease.
 64. The vaccine of any one of claims 25-63, whereinthe neutralizing antibody titer against hMPV in a subject followingadministration of a second dose of the vaccine is increased by 8-10 foldat 14 days post-administration of the second dose.
 65. The vaccine ofany one of claims 25-64, wherein the neutralizing antibody titer againsthPIV3 in a subject following administration of a second dose of thevaccine is increased by 4-10 fold at 14 days post-administration of thesecond dose.
 66. A method of preventing a lower respiratory humanmetapneumovirus and/or human parainfluenza virus 3 (hMPV/hPIV3)infection in elderly subjects, comprising: administering to a subjectwho is 65 years of age or older the vaccine of any one of claims 25-65in an effective amount to prevent a lower respiratory hMPV/hPIV3infection in the subject.
 67. A method of treating a lower respiratoryhuman metapneumovirus and/or human parainfluenza virus 3 (hMPV/hPIV3)infection in elderly subjects, comprising: administering to a subjectwho is 65 years of age or older and is infected with hMPV/hPIV3 thevaccine of any one of claims 25-65 in an effective amount to treat alower respiratory hMPV/hPIV3 infection in the subject.
 68. A method ofpreventing a lower respiratory human metapneumovirus and/or humanparainfluenza virus 3 (hMPV/hPIV3) infection in a child, comprising:administering to a subject who is 5 years of age or younger the vaccineof any one of claims 25-65 in an effective amount to prevent a lowerrespiratory hMPV/hPIV3 infection in the subject.
 69. A method oftreating a lower respiratory human metapneumovirus and/or humanparainfluenza virus 3 (hMPV/hPIV3) infection in a child, comprising:administering to a subject who is 5 years of age or younger and isinfected with hMPV/hPIV3 the vaccine of any one of claims 25-65 in aneffective amount to treat a lower respiratory hMPV/hPIV3 infection inthe subject.
 70. The method of claim 69, wherein the subject is between6 and 12 months of age.
 71. A method of preventing a lower respiratoryhuman metapneumovirus and/or human parainfluenza virus 3 (hMPV/hPIV3)infection in subjects having a pulmonary disease, comprising:administering to a subject having a pulmonary disease the vaccine of anyone of claims 25-65 in an effective amount to prevent a lowerrespiratory hMPV/hPIV3 infection in the subject.
 72. A method oftreating a lower respiratory human metapneumovirus and/or humanparainfluenza virus 3 (hMPV/hPIV3) infection in subjects having apulmonary disease, comprising: administering to a subject having apulmonary condition the vaccine of any one of claims 25-65 in aneffective amount to treat a lower respiratory hMPV/hPIV3 infection inthe subject.
 73. The method of claim 72, wherein the pulmonary conditionis associated with is Chronic Obstructive Pulmonary Disease (COPD),asthma, congestive heart failure or diabetes, or any combinationthereof.
 74. A method of preventing a lower respiratory humanmetapneumovirus and/or human parainfluenza virus 3 (hMPV/hPIV3)infection in immunocompromised subjects, comprising: administering to animmunocompromised subject the vaccine of any one of claims 25-65 in aneffective amount to prevent a lower respiratory hMPV/hPIV3 infection inthe subject.
 75. A method of treating a lower respiratory humanmetapneumovirus and/or human parainfluenza virus 3 (hMPV/hPIV3)infection in immunocompromised subjects, comprising: administering to animmunocompromised subject the vaccine of any one of claims 25-54 in aneffective amount to treat a lower respiratory hMPV/hPIV3 infection inthe subject.
 76. The method of any one of claims 66-75, wherein a singledose of the vaccine is administered to the subject.
 77. The method ofany one of claims 66-75, wherein a booster dose of the vaccine isadministered to the subject.
 78. The method of claim 77, wherein theefficacy of the vaccine against the hMPV/hPIV3 infection is at least 50%following administration of the booster dose of the vaccine.
 79. Themethod of claim 78, wherein the efficacy of the vaccine against thehMPV/hPIV3 infection is at least 60% following administration of thebooster dose of the vaccine.
 80. The method of claim 79, wherein theefficacy of the vaccine against the hMPV and/or hPIV3 infection is atleast 65% following administration of a single dose of the vaccine. 81.The method of claim 80, wherein the efficacy of the vaccine against thehMPV and/or hPIV3 infection is at least 70% following administration ofa single dose of the vaccine.
 82. The method of claim 81, wherein theefficacy of the vaccine against the hMPV and/or hPIV3 infection is atleast 75% following administration of a single dose of the vaccine. 83.The method of any one of claims 66-82, wherein the vaccine immunizes thesubject against hMPV/hPIV3 for up to 2 years.
 84. The method of any oneof claims 66-82, wherein the vaccine immunizes the subject againsthMPV/hPIV3 for more than 2 years.
 85. A vaccine, comprising: (a) 12.5μg-200 μg a human metapneumovirus (hMPV) ribonucleic acid (RNA)polynucleotide comprising the nucleic acid sequence identified by SEQ IDNO:4; and (b) 12.5 μg-200 μg a human parainfluenza virus 3 (hPIV3) RNApolynucleotide comprising the nucleic acid sequence identified by SEQ IDNO:5, wherein the RNA polynucleotides of (a) and (b) are formulated in alipid nanoparticle comprising a Compound 1 of Formula (I), aPEG-modified lipid, a sterol and a non-cationic lipid.
 86. The method ofclaim 85, wherein the efficacy of the vaccine against the hMPV/hPIV3infection is at least 50%, relative to control, following administrationof the booster dose of the vaccine.
 87. The vaccine of claim 86, whereinthe efficacy of the vaccine against the hMPV/hPIV3 infection is at least70%, relative to control following administration of a single dose ofthe vaccine.
 88. Use of the vaccine of any one of claims 25-65 in themanufacture of a medicament for use in a method of inducing an antigenspecific immune response to hMPV/hPIV3 in a subject, the methodcomprising administering to the subject the vaccine in an amounteffective to produce an antigen specific immune response to hMPV/hPIV3in the subject.
 89. A pharmaceutical composition for use in vaccinationof a subject comprising an effective dose of the vaccine of any one ofclaims 25-65, wherein the effective dose is sufficient to producedetectable levels of antigen as measured in serum of the subject at 1-72hours post administration.
 90. The pharmaceutical composition of claim89, wherein the cut off index of the antigen is 1-2.
 91. Apharmaceutical composition for use in vaccination of a subjectcomprising an effective dose of the vaccine of any one of claims 25-65,wherein the effective dose is sufficient to produce a 1,000-10,000neutralization titer produced by neutralizing antibody againsthMPV/hPIV3 antigen as measured in serum of the subject at 1-72 hourspost administration.
 92. A vaccine, comprising: (a) a humanmetapneumovirus (hMPV) ribonucleic acid (RNA) polynucleotide comprisingthe nucleic acid sequence identified by SEQ ID NO:4 or a nucleic acidsequence that is at least 95% identical to the nucleic acid sequenceidentified by SEQ ID NO:4; and (b) a human parainfluenza virus 3 (hPIV3)RNA polynucleotide comprising the nucleic acid sequence identified bySEQ ID NO:5 or a nucleic acid sequence that is at least 95% identical tothe nucleic acid sequence identified by SEQ ID NO:5, wherein the RNApolynucleotides of (a) and (b) are formulated in a lipid nanoparticlecomprising a cationic lipid, a PEG-modified lipid, a sterol and anon-cationic lipid.
 93. A vaccine, comprising: (a) a humanmetapneumovirus (hMPV) ribonucleic acid (RNA) polynucleotide encoded bya nucleic acid comprising the nucleic acid sequence identified by SEQ IDNO:1 or a nucleic acid sequence that is at least 95% identical to thenucleic acid sequence identified by SEQ ID NO:1; and (b) a humanparainfluenza virus 3 (hPIV3) RNA polynucleotide encoded by a nucleicacid comprising the nucleic acid sequence identified by SEQ ID NO:2 or anucleic acid sequence that is at least 95% identical to the nucleic acidsequence identified by SEQ ID NO:2, wherein the RNA polynucleotides of(a) and (b) are formulated in a lipid nanoparticle comprising a cationiclipid, a PEG-modified lipid, a sterol and a non-cationic lipid.
 94. Anengineered polynucleotide comprising the nucleic acid sequenceidentified by SEQ ID NO:2.
 95. An engineered polynucleotide comprisingthe nucleic acid sequence identified by SEQ ID NO:5.
 96. An engineeredpolypeptide comprising the amino acid sequence identified by SEQ IDNO:8.